Compositions containing inclusion complexes

ABSTRACT

The invention provides a composition containing particulate composite of a polymer and a therapeutic agent. The composition also contains a complexing agent. The polymer interacts with the complexing agent in a host-guest or a guest-host interaction to form an inclusion complex. A therapeutic composition of the invention may be used to deliver the therapeutic agent and to treat various disorders. Both the polymer of the particulate composite and the complexing agent may be used to introduce functionality into the therapeutic composition. The invention also relates to a method of preparing a composition. The method combines a therapeutic agent, a polymer having host or guest functionality, and a complexing agent having guest or host functionality to form the therapeutic composition. The complexing agent forms an inclusion complex with the polymer. The invention also relates to a method of delivering a therapeutic agent. According to the method, a therapeutically effective amount of a therapeutic composition of the invention is administered to a mammal (e.g. person or animal) in recognized need of the therapeutic. Also disclosed are compounds having the formula:

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/321,441, filed Dec. 28, 2005, now issued U.S. Pat. No. 7,807,198,which is a divisional of U.S. application Ser. No. 10/021,312, filedDec. 19, 2001, now issued U.S. Pat. No. 7,018,609, which claims thebenefit of U.S. provisional application Nos. 60/256,341, filed Dec. 19,2000; 60/256,344, filed Dec. 19, 2000; and 60/293,543, filed May 29,2001, each of which is incorporated by reference herein.

FIELD OF THE INVENTION

The invention relates compositions and methods used to delivertherapeutic agents. More particularly, the invention relates to acomposition containing a polymer, a therapeutic agent, and a complexingagent where the polymer interacts with the complexing agent in ahost-guest or a guest-host interaction to form an inclusion complex. Acomposition of the invention may be used to deliver a therapeutic agentin the treatment of various disorders.

BACKGROUND OF THE INVENTION

Cyclodextrins are cyclic polysaccharides containing naturally occurringD(+)-glucopyranose units in an α-(1, 4) linkage. The most commoncyclodextrins are alpha (α)-cyclodextrins, beta (β)-cyclodextrins andgamma (γ)-cyclodextrins which contain, respectively, six, seven or eightglucopyranose units. Structurally, the cyclic nature of a cyclodextrinforms a torus or donut-like shape having an inner apolar or hydrophobiccavity, the secondary hydroxyl groups situated on one side of thecyclodextrin torus and the primary hydroxyl groups situated on theother. Thus, using (β)-cyclodextrin as an example, a cyclodextrin isoften represented schematically as follows:

The side on which the secondary hydroxyl groups are located has a widerdiameter than the side on which the primary hydroxyl groups are located.The hydrophobic nature of the cyclodextrin inner cavity allows for theinclusion of a variety of compounds. (Comprehensive SupramolecularChemistry, Volume 3, J. L. Atwood et al., eds., Pergamon Press (1996);T. Cserhati, Analytical Biochemistry, 225:328-332 (1995); Husain et al.,Applied Spectroscopy, 46:652-658 (1992); FR 2 665 169).

Cyclodextrins have been used as a delivery vehicle of varioustherapeutic compounds by forming inclusion complexes with various drugsthat can fit into the hydrophobic cavity of the cyclodextrin or byforming non-covalent association complexes with other biologicallyactive molecules such as oligonucleotides and derivatives thereof. Forexample, U.S. Pat. No. 4,727,064 describes pharmaceutical preparationsconsisting of a drug with substantially low water solubility and anamorphous, water-soluble cyclodextrin-based mixture. The drug forms aninclusion complex with the cyclodextrins of the mixture. In U.S. Pat.No. 5,691,316, a cyclodextrin cellular delivery system foroligonucleotides is described. In such a system, an oligonucleotide isnoncovalently complexed with a cyclodextrin or, alternatively, theoligonucleotide may be covalently bound to adamantane which in turn isnon-covalently associated with a cyclodextrin.

Various cyclodextrin containing polymers and methods of theirpreparation are also known in the art. (Comprehensive SupramolecularChemistry, Volume 3, J. L. Atwood et al., eds., Pergamon Press (1996)).A process for producing a polymer containing immobilized cyclodextrin isdescribed in U.S. Pat. No. 5,608,015. According to the process, acyclodextrin derivative is reacted with either an acid halide monomer ofan α, β-unsaturated acid or derivative thereof or with an α,β-unsaturated acid or derivative thereof having a terminal isocyanategroup or a derivative thereof. The cyclodextrin derivative is obtainedby reacting cyclodextrin with such compounds as carbonyl halides andacid anhydrides. The resulting polymer contains cyclodextrin units asside chains off a linear polymer main chain.

U.S. Pat. No. 5,276,088 describes a method of synthesizing cyclodextrinpolymers by either reacting polyvinyl alcohol or cellulose orderivatives thereof with cyclodextrin derivatives or by copolymerizationof a cyclodextrin derivative with vinyl acetate or methyl methacrylate.Again, the resulting cyclodextrin polymer contains a cyclodextrin moietyas a pendant moiety off the main chain of the polymer.

A biodegradable medicinal polymer assembly with supermolecular structureis described in WO 96/09073 A1 and U.S. Pat. No. 5,855,900. The assemblycomprises a number of drug-carrying cyclic compounds prepared by bindinga drug to an α, β or γ-cyclodextrin and then stringing thedrug/cyclodextrin compounds along a linear polymer with thebiodegradable moieties bound to both ends of the polymer. Such anassembly is reportably capable of releasing a drug in response to aspecific biodegradation occurring in a disease. These assemblies arecommonly referred to as “necklace-type” cyclodextrin polymers.

However, there exists a need in the art for a more effective non-viraldelivery systems exhibiting properties such as, for example, increasedstability (e.g. under physiological conditions) and effective targetingabilities. This invention answers such a need.

SUMMARY OF THE INVENTION

The invention provides a composition containing of a polymer, atherapeutic agent and a complexing agent. The polymer interacts with thecomplexing agent in a host-guest and/or a guest-host interaction to forman inclusion complex. A composition of the invention may be used todeliver a therapeutic agent and in the treatment of various disorders.Both the polymer and the complexing agent may be used to introducefunctionality into the composition.

The invention provides a composition comprising a particulate compositeof a polymer and a therapeutic agent and an inclusion complex of thepolymer and the complexing agent. The polymer of the particulatecomposite may have host functionality and forms an inclusion complexwith a guest complexing agent.

Alternatively, at least one polymer of the particulate composite hasguest functionality and forms an inclusion complex with a hostcomplexing agent. In another embodiment the polymer or the complexingagent may have both host and guest functionalities which form inclusioncomplexes. This allows multiple complexing agents to form inclusioncomplexes and thereby become associated with the therapeuticcomposition. This also allows for multiple functionalites to beintroduced into the therapeutic composition of the invention.

The invention also relates to a method of preparing a therapeuticcomposition. The method combines a therapeutic agent, a polymer havinghost or guest functionality, and a complexing agent having guest or hostfunctionality to form the therapeutic composition. The complexing agentforms an inclusion complex with the polymer.

The invention also relates to a method of delivering a therapeuticagent. According to the method, a therapeutically effective amount of acomposition of the invention is administered to a mammal (e.g. person oranimal) in recognized need of the therapeutic agent. Thus, the inventionprovides for treatment of a disease using a composition of the inventionto deliver an appropriate therapeutic agent.

BRIEF DESCRIPTION OF DRAWINGS

In the Figures depicting various embodiments of the invention, compound12 is also designated as βCDP6. Composites having a nucleic acid and acationic polymer in the particulate composite are identified aspolyplexes. The brief descriptions of the figures are as follows.

FIG. 1. Structures of various adamantane-PEG Molecules

FIG. 2. Hydrodynamic diameter of GALA and GALA-Ad modified compositions,Example 30.

FIG. 3. Zeta Potential of GALA and GALA-Ad modified compositions,Example 32.

FIG. 4. Uptake of GALA-Ad and GALA modified compositions by BHK-21cells, Example 31.

FIG. 5. Uptake of GALA-Ad and GALA modified polyplex polyplexcompositions by HUH-7 cells, Example 33.

FIG. 6. Luciferase transfection of BHK-21 cells with β-cyclodextrin-DMScopolymer 12-based compositions modified with GALA and GALA -Ad, Example34.

FIG. 7. Toxicity of GALA and GALA-Ad modified polyplexes to BHK-21cells, Example 35.

FIG. 8. Scheme for post-DNA-complexation pegylation by grafting, Example39.

FIG. 9. Particle sizes of PEI and 12 particulate composites and polyplexpolyplex compositions during post-DNA-complexation, Example 39.

FIG. 10. Stabilization of polyplex compositions by pegylation, Example40.

FIG. 11. Co-delivery of 12 polyplexes with PEG₃₄₀₀-FITC, Example 42.

FIG. 12. Structure of Lactose −12, Example 37.

FIG. 13. Transfection of 12 and LAC-CDP6 polyplexes to HUH-7 cells,Example 43.

FIG. 14. Schematic of Experimental Protocal, Example 47.

FIG. 15. Particle Diameters, Example 47.

FIG. 16. DNA loss due to complex precipitation, Example 47.

FIG. 17. Inclusion Complexes to Modify 12/DNA Composite, Example 48.

FIG. 18. Transfection of Modified Polyplexes to HepG2 cells, Example 49.

FIG. 19. Competitive Displacement Experiments, Example 52.

FIG. 20. Synthesis of Adamantane-PEG-Transferrin (Ad-PEG-Tf), Example55.

FIG. 21. Iron loading for transferrin, Example 55.

FIG. 22. Binding Affinity Transferrin-PEG-Ad, Example 55.

FIG. 23. Transferrin coupling via Lysine groups, Example 56.

FIG. 24. Binding affinity of Transferrin-PEG-AD to transferrin receptorson PC3 cells, Example 57.

FIG. 25. Zeta potential variation and particle size as a function ofparticle modification in transferrin and PEG-modified polyplexes,Example 58.

FIG. 26. Zeta potential measurements, Ad-anionic-PEG, Example 62.

FIG. 27. Stability Measurements, Example 62.

FIG. 28. Addition of increasing transferrin complexing agent, Example62.

FIG. 29. Synthesis of Histidylated 12.

FIG. 30. pH-sensitive Polymers for Endosomal Escape (Synthesis ofsecondary amine containing polymers).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a composition that employs inclusion complexesto deliver therapeutic agents. Inclusion complexes are molecularcompounds having the characteristic structure of an adduct, in which oneof the compounds (host molecule) spatially encloses at least part ofanother. The enclosed compound (guest molecule) is situated in thecavity of the host molecule without affecting the framework structure ofthe host. It is a characteristic feature of an inclusion complex thatthe size and shape of the available cavity remain most often practicallyunaltered, apart from a slight deformation. A “host” may be any hostcompound or molecule known in the art. Examples of suitable “hosts”include, but are not limited to, cyclodextrins, carcerands, cavitands,crown ethers, cryptands, cucurbiturils, calixarenes, spherands, and thelike. Examples of inclusion guests suitable for the complexing agentsinclude those known in the art such as, but not limited to, adamantane,diadamantane, naphthalene, and cholesterol.

Cyclodextrins are a preferred host, able to interact with a greatvariety of ionic and molecular species and the resulting inclusioncompounds belonging to the class of “host-guest” compelexes. For therealization of the host-guest relationship several requirements must bemet; one of them is that the binding sites of the host and guestmolecules should be complementary in the stereoelectronic sense.Cyclodextrins are capable of forming inclusion complexes with compoundshaving a size compatible with the dimensions of the cavity. The extentof complex formation depends, however, also on the polarity of the guestmolecule. Complex formation with molecules significantly larger than thecavity may also be possible in such a way that only certain groups orside chains penetrate into the carbohydrate channel. See J. Szejtli,Akademiai Kiado, Cyclodextrins and their inclusion complexes, Budapest,1982.

A composition of the invention contains at least one polymer and atleast one therapeutic agent, generally in the forth of a particulatecomposite of a polymer and therapeutic agent. The therapeuticcomposition also contains one or more complexing agents. At least onepolymer of the particulate composite interacts with the complexing agentin a host-guest or a guest-host interaction to form an inclusion complexbetween the polymer and the complexing agent. The polymer and, moreparticularly the complexing agent may be used to introduce functionalityinto a composition of the invention. In one embodiment, at least onepolymer of the particulate composite has host functionality and forms aninclusion complex with a complexing agent having guest functionality. Inanother embodiment, at ieast one polymer of the particulate compositehas guest functionality and forms an inclusion complex with a complexingagent having host functionality. In a further embodiment a polymer ofthe particulate composite may contain both host and guestfunctionalities and form inclusion complexes with guest complexingagents and host complexing agents.

1. The Particulate Composite

A particulate composite of a therapeutic agent and a polymer is acombination or integration of a therapeutic agent and a polymer. Theparticulate composite is an associated structure comprising one or moretherapeutic agents within a multi-dimensional polymer network. A singlepolymer or a mixture of polymers may be used. In addition to beingcapable of forming the multi-dimensional polymer network of theparticulate composite, at least one polymer of the composite, asdiscussed below, carries host and/or guest functionality capable offorming inclusion complexes with one or more complexing agents.

A. The Polymer

Any type of polymer capable of forming a particulate composite with atherapeutic agent and having host and/or guest functionality may be usedin the composition of the invention. The polymer may be a linear orbranched polymer. The polymer may be a homopolymer or a co-polymer. If aco-polymer is used, the co-polymer may be a random copolymer or abranched co-polymer. Preferably the polymer is water-dispersible andmore preferably water soluble. For example, suitable polymers include,but are not limited to polysaccharides, polyesters, polyamides,polyethers, polycarbonates, polyacrylates, etc. For therapeuticpharmaceutical uses, the polymer should have a low toxicity, profile andpreferably are not toxic or cyclotoxic. As discussed below, a preferredpolymer for use in a composition of the invention is acyclodextrin-based polymer. Water soluble linear cyclodextrincopolymers, described below, having molecular weights in the range of3,000 to 100,000 are preferred and those having molecular weights of3,000 to 50,000 are particularly preferred.

According to the invention, the polymer in the particulate composite maybe a single polymer or as a mixture of two or more polymers, which maybe the same or different polymers. Each polymer of the particulatecomposite may further contain or may be further modified to contain acrosslinking group through which association of the polymers to form theparticulate composite may be achieved.

At least one polymer of the particulate composite is a polymer capableof forming an inclusion complex. A “polymer capable of inclusion complexformation” may be any polymer capable of one or more host-guestassociations via nonbonding interactions (e.g. van der Waals forces,hydrogen bonding, dipole-dipole interactions, ion-paring, soluophobicinteractions, etc.) with another compound (the complexing agent) orsubstituent on a compound. In other words, at least one polymer has hostor guest functionality to form an inclusion complex with a complexingagent or a substituent on the complexing agent. The host or guestfunctionality may be part of the polymer backbone or may be present as asubstituent or in a pendant or branched chain. An example of a polymerhaving host functionality in the polymer backbone is a linearcyclodextrin polymer as described below. An example of a polymer havingguest functionality not as part of the polymer backbone would be apolymer having pendant adamantane groups. Other examples of suitable“hosts” which may be employed with the polymer include, but are notlimited to, carceronds, cavitands, crown ethers, cryptands,cucurbiturils, calixarenes, spherands and the like. Examples ofinclusion guests suitable for such hosts include those known in the artsuch as, but not limited to, adamantane, diadamantane, naphthalene, andcholesterol.

In a preferred embodiment, a polymer may contain different types of hostor guest functionalities or the polymer may contain both host and guestfunctionality. This allows even greater flexibility for differentinclusion complexes to be formed on a given polymer. Having multiplehost, multiple guest, or both host and guest functionalities on the samepolymer increases the variety of functionality which may introduced intoa therapeutic composition of the invention via the inclusion complex.

As a result of the host-guest association, the polymer interacts withthe complexing agent to form an inclusion complex. Preferably, as aresult of the nonbonding interaction or association, the resultinginclusion complex exhibits binding constants of about >10², preferably,about >10³, and more preferably, about >10⁴. Typically, bindingconstants will range from about 10²-0⁶.

A polymer of the particulate composite may be modified with one or moreligands. The ligand may be introduced upon or after formation of theparticulate composite via ligand modification of the therapeutic agentand/or the polymer of the particulate composite. The ligand may be anyligand that allows for targeting and/or binding to a desired cell. Aswould be understood by one of skill in the art, targeting and binding toa cell may include cell receptor attachment which in turn may lead toreceptor mediated endocytosis. If two or more ligands are attached, theligands may be the same or different. Examples of suitable ligandsinclude, but are not limited to, vitamins (e.g. folic acid), proteins(e.g. transferrin, and monoclonal antibodies), monosaccharides (e.g.galactose), peptides, and polysaccharides. The choice of ligand, as oneof ordinary skill appreciates, may vary depending upon the type ofdelivery desired. As another example, the ligand may be membranepermeabilizing or membrane permeable agent such as the TAT protein fromHTV-1. The TAT protein is a viral transcriptional activation that isactively imported into the cell nucleous. Torchilin, V. P. et al, PNAS.98, 8786-8791, (2001).

In a preferred embodiment of the invention, at least one of the polymersof the particulate composite is a substantially linear polymer havinghost and/or guest functionality capable of forming an inclusion complex.A substantially linear polymer may be prepared by any means known in theart. The polymer may be prepared from a suitable monomer capable ofinclusion complex formation or a mixture of monomers of which at leastone has host or guest functionality. The host or guest functionality maybe within the polymer chain, pendant (or branched) to the polymer chain,or present as an end-group. Alternatively, after the polymer is formed,it may be further modified to add host and/or guest funtionality, asdiscussed above, to form a substantially linear polymer capable ofinclusion complex formation. The substantially linear polymer may be ablock co-polymer where the blocks introduce properties such as hostfunctionality, water-dispersiblility and/or water-solubility. Examplesof such blocks include, for example, linear polyethyleneimine (PEI), alinear cyclodextrin-containing polymer,bis(2-aminoethyl)-1,3-propanediamine (AEPD), and N₂, N₂, N₃,N3-(3′-PEG₅₀₀₀-aminopropane)-bis(2-aminoethyl)-1,3-propanediammoniumdi-trifluoroacetate (AEPD-PEG).

In another preferred embodiment, the polymer used to form theparticulate composite is a cyclodextrin-containing polymer, morepreferably a substantially linear cyclodextrin polymer as describedbelow. The polymer may also be a polyethyleneimine (PEI) or a polymerhaving pendant cyclodextrins. A linear cyclodextrin copolymer is apolymer containing cyclodextrin moieties as an integral part of itspolymer backbone. Polymers having pendant cyclodextrin moieties not apart of the main polymer chain but rather attached off the polymerbackbone may also be used in the compositions of the invention. A linearcyclodextrin-containing polymer may be any linear polymer containing atleast one cyclodextrin moiety as part of the polymer backbone. Thecyclodextrin-containing polymer is preferably water-soluble. Morepreferably, the linear cyclodextrin-containing polymer is a linearcyclodextrin copolymer or a linear oxidized cyclodextrin copolymer, eachas described below. The cyclodextrin groups within the polymer providehost functionality to the polymer allowing it to form inclusioncomplexes. The substantially linear polymer capable of inclusion complexformation may further contain or may be further modified to contain anadditional functional group (e.g. thiol group).

Linear Cyclodextrin-Containing Polymers

A linear cyclodextrin copolymer which can be used to form theparticulate composite contains substituted or unsubstituted,cyclodextrin moieties bifunctionally bound in the linear copolymerbackbone, through the number 2, 3, or 6 position of at least oneglucopyranose ring of the cyclodextrin, to divalent moieties linking thecyclodextrins of the linear cyclodextrin polymer. As described in WO00/01734 such a linear cyclodextrin copolymer has a repeating unit offormula Ia, Ib, (below) or a combination thereof. Linear cyclodextincopolymers, their preparation and properties, are also described inGonzalez, H., Hwang, S, and Davis, M. (1999) New class of polymers forthe delivery of macromolecular therapeutics. Bioconjugate Chem, 10,1068-1074 and Hwang, S., Bellocq, N. and Davis, M. (2001) Effects ofStructure of Beta-Cyclodextrin-Containing Polymers on Gene Delivery.Bioconjugate Chem, 12(2), 280-290, both of which are incorporated hereby reference.

In formulae Ia and Ib, C is a substituted or unsubstituted cyclodextrinmonomer and A is a cpmonomer bound, i.e. covalently bound, tocyclodextrin C. Polymerization of a cyclodextrin monomer C precursorwith a comonomer A precursor results in a linear cyclodextrin copolymer.Within a single linear cyclodextrin copolymer, the cyclodextrin monomerC unit may be the same or different and, likewise, the comonomer A maybe the same or different.

A cyclodextrin monomer precursor may be any cyclodextrin or derivativethereof known in the art. As discussed above, a cyclodextrin is definedas a cyclic polysaccharide most commonly containing six to eightnaturally occurring D(+)-glucopyranose units in an a-(1, 4) linkage.Preferably, the cyclodextrin monomer precursor is a cyclodextrin havingsix, seven and eight glucose units, i.e., respectively, an alpha(α)-cyclodextrin, a beta (p)-cyclodextrin and a gamma (γ)-cyclodextrin.A cyclodextrin derivative may be any substituted cyclodextrin known inthe art where the substituent does not interfere with copolymerizationwith comonomer A precursor as described below. A cyclodextrin derivativemay be neutral, cationic or anionic. Examples of suitable substituentsinclude, but are not limited to, hydroxyalkyl groups, such as, forexample, hydroxypropyl, hydroxyethyl; ether groups, such as, forexample, dihydroxypropyl ethers, methyl-hydroxyethyl ethers,ethyl-hydroxyethyl ethers, and ethyl-hydroxypropyl ethers; alkyl groups,such as, for example, methyl; saccharides, such as, for example,glucosyl and maltosyl; acid groups, such as, for example, carboxylicacids, phosphorous acids, phosphinous acids, phosphonic acids,phosphoric acids, thiophosphonic acids, and sulfonic acids; imidazolegroups; sulfate groups; and protected thiol groups.

A cyclodextrin monomer precursor may be further chemically modified(e.g. halogenated, aminated) to facilitate or affect copolymerization ofthe cyclodextrin monomer precursor with a comonomer A precursor, asdescribed below. Chemical modification of a cyclodextrin monomerprecursor allows for polymerization at only two positions on eachcyclodextrin moiety, i.e. the creation of a bifunctional cyclodextrinmoiety. The numbering scheme for the C1-C6 positions of eachglucopyranose ring is as follows:

In a preferred embodiment, polymerization occurs at two of any C2, C3and C6 position, including combinations thereof, of the cyclodextrinmoiety. For example, one cyclodextrin monomer precursor may bepolymerized at two C6 positions while another cyclodextrin monomerprecursor may be polymerized at a C2 and a C6 position of thecyclodextrin moiety. Using β-cyclodextrin as an example, the letteringscheme for the relative position of each glucopyranose ring in acyclodextrin is as follows:

In a preferred embodiment of a linear cyclodextrin copolymer, thecyclodextrin monomer C has the following general formula (II):

In formula (II), n and m represent integers which, along with the othertwo glucopyranose rings, define the total number of glucopyranose unitsin the cyclodextrin monomer. Formula (II) represents a cyclodextrinmonomer which is capable of being polymerized at two C6 positions on thecyclodextrin unit. Examples of cyclodextrin monomers of formula (II)include, but are not limited to, 6^(A), 6^(B)-dideoxy-α-cyclodextrin(n=0, m=4), 6^(A), 6^(c)-dideoxy-α-cyclodextrin (n=1, m=3), 6^(A),6^(D)-dideoxy-α-cyclodextrin (n=2, m=2), 6^(A),6^(B)-dideoxy-β-cyclodextrin (n=0, m=5), 6^(A),6^(c)-dideoxy-β-cyclodextrin (n=1, m=4), 6^(A),6^(D)-dideoxy-β-cyclodextrin (n=2, m=3), 6^(A),6^(B)-dideoxy-γ-cyclodextrin (n=0, m=6), 6^(A),6^(c)-dideoxy-Y-cyclodextrin (n-1, m=5), 6^(A),6^(D)-dideoxy-γ-cyclodextrin (n=2, m=4), and 6^(A),6^(E)-dideoxy-y-cyclodextrin (n=3, m=3).

In another preferred embodiment of a linear cyclodextrin copolymer cancontain a glucose-ring-opened cyclodextrin monomer C unit where one ormore of the glucopyranose rings of the cyclodextrin has been openedwhile maintaining the cyclodextrin ring system. General formula (III),below, depicts a glucopyranose-ring-opened cyclodextrin with ringopening at the C2, C3 positions.

In formula (TH) p varies from 5-7. In formula (TH), at least one ofD(+)-glucopyranose units of a cyclodextrin monomer has undergone ringopening to allow for polymerization at a C2 and a C3 position of thecyclodextrin unit. Cyclodextrin monomers of formula (EI) such as, forexample, 2^(A), 3^(A)-âamino-2^(A), 3^(A)-dideoxy-β-cyclodextrin and2^(A), 3^(A)-dialdehyde-2^(A), 3^(A)-dideoxy-β-cyclodextrin arecommercially available from Carbomer of Westborough, Mass. Examples ofcyclodextrin monomers of formula (III) include, but are not limited to,2^(A), 3^(A)-dideoxy-2^(A), 3^(A)-dihydro-α-cyclodextrin, 2^(A),3^(A)-dideoxy-2^(A), 3^(A)-dihydro-β-cyclodextrin, 2^(A),3^(A)-dideoxy-2^(A), 3^(A)-dihydro-γ-cyclodextrin, commonly referred toas, respectively, 2,3-dideoxy-α-cyclodextrin,2,3-dideoxy-β-cyclodextrin, and 2,3-dideoxy-γ-cyclodextrin.

A comonomer A precursor may be any straight chain or branched, symmetricor asymmetric compound which upon reaction with a cyclodextrin monomerprecursor, as described above, links two cyclodextrin monomers together.Preferably, a comonomer A precursor is a compound containing at leasttwo crosslinking groups through which reaction and thus linkage of thecyclodextrin monomers can be achieved. Examples of possible crosslinkinggroups, which may be the same or different, terminal or internal, ofeach comonomer A precursor include, but are not limited to, amino, acid,ester, imidazole, and acyl halide groups and derivatives thereof. In apreferred embodiment, the two crosslinking groups are the same andterminal. Upon copolymerization of a comonomer A precursor with acyclodextrin monomer precursor, two cyclodextrin monomers may be linkedtogether by joining the primary hydroxyl side of one cyclodextrinmonomer with the primary hydroxyl side of another cyclodextrin monomer,by joining the secondary hydroxyl side of one cyclodextrin monomer withthe secondary hydroxyl side of another cyclodextrin monomer, or byjoining the primary hydroxyl side of one cyclodextrin monomer with thesecondary hydroxyl side of another cyclodextrin monomer. Accordingly,combinations of such linkages may exist in the final copolymer.

Both the comonomer A precursor and the comonomer A of the finalcopolymer may be neutral, cationic (e.g. by containing protoriatedgroups such as, for example, quaternary ammonium groups) or anionic(e.g. by containing deprotonated groups, such as, for example, sulfate,phosphate or carboxylate anionic groups). The counterion of a chargedcomonomer A precursor or comonomer A may be any suitable counteranion orcountercation (e.g. the counteranion of a cationic comonomer A precursoror comonomer A may be a halide (e.g chloride) anion). The charge ofcomonomer A of the copolymer may be adjusted by adjusting pH conditions.

Examples of suitable comonomer A precursors include, but are not limitedto, cystamine, 1,6-diaminohexane, diimidazole, dithioimidazole,spermine, dithiospermine, dihistidine, dithiohistidine, succinimide(e.g. dithiobis(succinimidyl propionate) (DSP) and disuccinimidylsuberate (DSS)), and imidates (e.g. dimethyl 3,3′-dithiobispropion-imidate (DTBP)). Copolymerization of a comonomer Aprecursor with a cyclodextrin monomer precursor leads to the formationof a linear cyclodextrin copolymer containing comonomer A linkages ofthe following general formulae: —HNC(O)(CH₂)_(x)C(O)NH—,—HNC(O)(CH₂)_(x)SS(CH₂)_(x)C(O)NH—, —⁺H₂N(CH₂)_(x)SS(CH₂)_(x)NH₂⁺—HNC(O)(CH₂CH₂O)_(n)CH₂CH₂C(O)NH—, ═NNHC(O)(CH₂CH₂O)_(x)CH₂CH₂C(O)NHN═,—⁺H₂NCH₂(CH₂CH₂O)_(n)CH₂CH₂CH₂NH₂ ⁺—,—HNC(O)(CH₂CH₂O)_(n)CH₂CH₂SS(CH₂CH₂O)_(x)CH₂CH₂C(O)NH—, —HNC(NH₂⁺)(CH₂CH₂O)_(x)CH₂CH₂C(NH₂ ⁺)NfH—, —SCH₂CH₂NHC(NH₂ ⁺)(CH₂)_(x)C(NH₂⁺)NHCH₂CH₂S—, —SCH₂CH₂NHC(NH₂ ⁺)(CH₂)_(x)SS(CH₂)_(x)C(NH₂ ⁺)NHCH₂CH₂S—,—SCH₂CH₂NHC(NH₂ ⁺)CH₂CH₂(OCH₂CH₂)_(x)C(NH₂ ⁺)NHCH₂CH₂S—,

In the above formulae, x=1-50, and y+z=x. Preferably, x=1-30. Morepreferably, x=1-20. In a preferred embodiment, comonomer A contains abiodegradable linkage such as a disulfide linkage. Comonomer A may alsoinclude acid-labile containing functionality such as esters and othersuch acid labile groups known to those skilled in the art.

In another preferred embodiment, the comonomer A precursor and hence thecomonomer A may be selectively chosen in order to achieve a desiredapplication. For example, to deliver small molecule therapeutic agents,a charged polymer may not be necessary and the comonomer A may be orcontain a hydrophilic group such as a polyethylene glycol group furtherenhancing water solubility. For polypeptide therapeutic agents such asDNA or proteins, the comonomer A preferably carries a cationic chargeincreasing the ability of the linear cyclodextrin copolymer to form aparticulate composite with the polypeptide therapeutic agent. It is alsounderstood that a linear cyclodextrin copolymer may contain a mixture ofcomonomer A groups.

A linear cyclodextrin copolymer may be prepared by copolymerizing acyclodextrin monomer precursor disubstituted with an appropriate leavinggroup with a comonomer A precursor capable of displacing the leavinggroups. The leaving group, which may be the same or different, may beany leaving group known in the art which may be displaced uponcopolymerization with a comonomer A precursor.

A linear cyclodextrin copolymer may be prepared by iodinating acyclodextrin monomer precursor to form a diiodinated cyclodextrinmonomer precursor and copolymerizing the diiodinated cyclodextrinmonomer precursor with a comonomer A precursor to form a linearcyclodextrin copolymer having a repeating unit of formula Ia, Ib, or acombination thereof, each as described above.

Another method of preparing a linear cyclodextrin iodinates acyclodextrin monomer precursor as described above to form a diiodinatedcyclodextrin monomer precursor of formula IVa, IVb, IVc or a mixturethereof:

The diiodinated cyclodextrin may be prepared by any means known in theart (see, e.g., Tabushi et al. J. Am. Chem. 106, 5267-5270 (1984);Tabushi et al. J. Am. Chem. 106, 4580-4584 (1984)). For example,β-cyclodextrin may be reacted with biphenyl-4,4′-disulfonyl chloride inthe presence of anhydrous pyridine to form a biphenyl-4, 4′-disulfonylchloride capped β-cyclodextrin which may then be reacted with potassiumiodide to produce diiodo-β-cyclodextrin. The cyclodextrin monomerprecursor is iodinated at only two positions. By copolymerizing thediiodinated cyclodextrin monomer precursor with a comonomer A precursor,as described above, a linear cyclodextrin polymer having a repeatingunit of formula Ia, Ib, or a combination thereof, also as describedabove, may be prepared. If appropriate, the iodine or iodo groups may bereplaced with other known leaving groups.

The iodo groups or other appropriate leaving group may be displaced witha group that permits reaction with a comonomer A precursor, as describedabove. For example, a diiodinated cyclodextrin monomer precursor offormula IVa, IVb, IVc or a mixture thereof may be aminated to form adiaminated cyclodextrin monomer precursor of formula Va, Vb, Vc or amixture thereof:

The diaminated cyclodextrin monomer precursor may be prepared by anymeans known in the art (see, e.g., Tabushi et al. Tetrahedron Lett.18:1527-1530 (1977); Mungall et al., J. Org. Chem. 1659-1662 (1975)).For example, a diiodo-β-cyclodextrin may be reacted with sodium azideand then reduced to form a diamino-β-cyclodextrin. The cyclodextrinmonomer precursor is aminated at only two positions. The diaminatedcyclodextrin monomer precursor may then be copolymerized with acomonomer A precursor, as described above, to produce a linearcyclodextrin copolymer having a repeating unit of formula Ia, Ib, or acombination thereof, also as described above. However, the aminofunctionality of a diaminated cyclodextrin monomer precursor need not bedirectly attached to the cyclodextrin moiety. Alternatively, the aminofunctionality may be introduced by displacement of the iodo or otherappropriate leaving groups of a cyclodextrin monomer precursor withamino group containing moieties such as, for example, —SCH₂CH₂NH₂, toform a diaminated cyclodextrin monomer precursor of formula Vd, Ve, Vf,Vg, Vh and Vi or a mixture thereof:

A linear cyclodextrin copolymer may also be prepared by reducing alinear oxidized cyclodextrin copolymer, as described below. This methodmay be performed as long as the comonomer A does not contain a reduciblemoiety or group such as, for example, a disulfide linkage.

A linear cyclodextrin copolymer may be oxidized so as to introduce atleast one oxidized cyclodextrin monomer into the copolymer such that theoxidized cyclodextrin monomer is an integral part of the polymerbackbone. A linear cyclodextrin copolymer which contains at least oneoxidized cyclodextrin monomer is defined as a linear oxidizedcyclodextrin copolymer. A linear oxidized cyclodextrin, then, hassubstituted or unsubstituted, cyclodextrin moieties bifunctionally boundin the linear copolymer backbone, through the number 2, 3, or 6 positionof at least one glucopyranose ring of the cyclodextrin, to bifunctionalmoieties, comomner A moieites, linking the cyclodextrins of the linearcyclodextrin polymer and wherein a glucopyranose ring of a cyclodextrinmoiety is oxidized. The cyclodextrin monomer may be oxidized on eitherthe secondary or primary hydroxyl side of the cyclodextrin moiety. Ifmore than one oxidized cyclodextrin monomer is present in a linearoxidized cyclodextrin copolymer, the same or different cyclodextrinmonomers oxidized on either the primary hydroxyl side, the secondaryhydroxyl side, or both may be present. For illustration purposes, alinear oxidized cyclodextrin copolymer with oxidized secondary hydroxylgroups has, for example, at least one unit of formula VIa or VIb:

In formulae VIa and VIb, C is a substituted or unsubstituted oxidizedcyclodextrin monomer and A is a comonomer bound, i.e. covalently bound,to the oxidized cyclodextrin C. Also in formulae VIa and VIb, oxidationof the secondary hydroxyl groups leads to ring opening of thecyclodextrin moiety and the formation of aldehyde groups.

A linear oxidized cyclodextrin copolymer may be prepared by oxidation ofa linear cyclodextrin copolymer as discussed above. Oxidation of alinear cyclodextrin copolymer may be accomplished by oxidationtechniques known in the art. (Hisamatsu et al., Starch 44:188-191(1992)). Preferably, an oxidant such as, for example, sodium periodateis used. It would be understood by one of ordinary skill in the art thatunder standard oxidation conditions that the degree of oxidation mayvary or be varied per copolymer. Thus in one embodiment, a linearoxidized copolymer may contain one oxidized cyclodextrin monomer. Inanother embodiment, substantially all to all cyclodextrin monomers ofthe copolymer would be oxidized.

Another method of preparing a linear oxidized cyclodextrin copolymerinvolves the oxidation of a diiodinated or diaminated cyclodextrinmonomer precursor, as described above, to form an oxidized diiodinatedor diaminated cyclodextrin monomer precursor and copolymerization of theoxidized diiodinated or diaminated cyclodextrin monomer precursor with acomonomer A precursor. In a preferred embodiment, an oxidizeddiiodinated cyclodextrin monomer precursor of formula VIIa, VIIb, VIIc,or a mixture thereof:

An oxidized cyclodextrin monomer may be prepared by oxidation of adiiodinated cyclodextrin monomer precursor of formulae IVa, IVb, IVc, ora mixture thereof, as described above. In another embodiment, anoxidized diaminated cyclodextrin monomer precursor of formula VIIIa,VIIIb, VIIIc or a mixture thereof.

may be prepared by animation of an oxidized diiodinated cyclodextrinmonomer precursor of formulae VIIa, VIIb, VIIc, or a mixture thereof, asdescribed above.

In still another embodiment, an oxidized diaminated cyclodextrin monomerprecursor of formula IXa, IXb, ac, IXd, IXe, IXf, or a mixture thereof

may be prepared by displacement of the iodo or other appropriate leavinggroups of an oxidized cyclodextrin monomer precursor disubstituted withan iodo or other appropriate leaving group with the amino groupcontaining moiety SCH₂CH₂NH₂.

Alternatively, an oxidized diiodinated, dicarboxylic acid, or diaminatedcyclodextrin monomer precursor, as described above, may be prepared byoxidizing a cyclodextrin monomer precursor to form an oxidizedcyclodextrin monomer precursor and then diiodinating and/or diaminatingthe oxidized cyclodextrin monomer, as described above. The amine groupsof any diaminated oxidized cyclodextrin monomers may be in theirprotected form to avoid unwanted side reactions. As discussed above, thecyclodextrin moiety may be modified with other leaving groups other thaniodo groups and other amino group containing functionalities. Theoxidized diiodinated or diaminated cyclodextrin monomer precursor maythen be copolymerized with a comonomer A precursor to form a linearoxidized cyclodextrin copolymer.

A linear cyclodextrin copolymer or a linear oxidized cyclodextrincopolymer terminates with at least one comonomer A precursor orhydrolyzed product of the comonomer A precursor. As a result oftermination of the cyclodextrin copolymer with at least one comonomer Aprecursor, a free derivatizing group, as described above, exists perlinear cyclodextrin copolymer or per linear oxidized cyclodextrincopolymer. For example, the derivatizing group may be an acid group or aderivatizing group that may be hydrolyzed to an acid group. According tothe invention, the derivatizing group may be further chemically modifiedas desired to enhance the properties of the cyclodextrin copolymer, suchas, for example, colloidal stability and transfection efficiency. Forexample, the derivatizing group may be modified by reaction with PEG toform a PEG terminated cyclodextrin copolymer to enhance colloidalstability or with histidine or imidazole acetic acid to form animidazolyl terminated cyclodextrin copolymer to enhance intracellular(e.g. endosomal release) and transfection efficiency. See FIGS. 29 and30.

Further chemistry may be performed on the cyclodextrin copolymer throughthe modified derivatizing group. For example, the modified derivatizinggroup may be used to extend a polymer chain by linking a linearcyclodextrin copolymer or linear oxidized cyclodextrin copolymer to thesame or different cyclodextrin copolymer or to a non-cyclodextrinpolymer. The polymer to be added on may be the same or different linearcyclodextrin copolymer or linear oxidized cyclodextrin copolymer whichmay also terminate with a comonomer A precursor for furthermodification.

Alternatively, at least two of the same or different linear cyclodextrincopolymers or linear oxidized cyclodextrin copolymers containing aterminal derivatizing group or a terminal modified derivatizing group,as described above, may be reacted and linked together through thefunctional or modified derivatizing group. Preferably, upon reaction ofthe functional or modified derivatizing groups, a degradable moiety suchas, for example, a disulfide linkage is formed. For example,modification of the terminal derivatizing group with cysteine may beused to produce a linear cyclodextrin copolymer or linear oxidizedcyclodextrin copolymer having a free thiol group. Reaction with the sameor different cyclodextrin copolymer also containing a free thiol groupwill form a disulfide linkage between the two copolymers. The functionalor modified derivatizing groups may be selected to offer linkagesexhibiting different rates of degradation (e.g. via enzymaticdegradation) and thereby provide, if desired, a time release system fora therapeutic agent. The resulting polymer may be crosslinked, asdescribed herein. A therapeutic agent, as described herein, may be addedprior to or post crosslinking of the polymer. A ligand may also be boundto the cyclodextrin copolymer through the modified derivatizing group.For example, a linear cyclodextrin copolymer or linear oxidizedcyclodextrin copolymer may be modified with a ligand attached to thecyclodextrin copolymer. The ligand may be attached to the cyclodextrincopolymer through the cyclodextrin monomer C or comonomer A. Preferably,the ligand is attached to a cyclodextrin moiety of the cyclodextrincopolymer. See WO 00/01734, incorporated here by reference.

Branched Cyclodextrin-Containing Polymers

The polymer of the particulate composite having host and/or guestfunctionality may also be a substantially branched polymer such as, forexample, branched polyethyleneimine (PEI) or a branchedcyclodextrin-containing polymer, preferably, a branchedcyclodextrin-containing polymer. A branched cyclodextrin-containingpolymer may be any water-soluble branched polymer containing at leastone cyclodextrin moiety which may be a part of the polymer backboneand/or pendant from the polymer backbone. A branchedcyclodextrin-containing polymer is a branched cyclodextrin copolymer ora branched oxidized cyclodextrin copolymer. A branched cyclodextrincopolymer or a branched oxidized cyclodextrin copolymer is,respectively, a linear cyclodextrin copolymer or a linear oxidizedcyclodextrin copolymer, as described above, from which a subordinatechain is branched. The branching subordinate chain may be any saturatedor unsaturated, linear or branched hydrocarbon chain. The branchingsubordinate chain may further contain various derivatizing groups orsubstituents such as, for example, hydroxyl, amino, acid, ester, amido,keto, formyl, and nitro groups. The branching subordinate chain may alsocontain a cyclodextrin or other host or guest functional moiety. Thebranching subordinate chain may also be modified with a ligand. Suchligand modification includes, but is not limited to, attachment of aligand to a cyclodextrin moiety in the branching subordinate chain.

Preferably, the branched cyclodextrin-containing polymer is a branchedcyclodextrin copolymer or a branched oxidized cyclodextrin copolymer ofwhich the branching subordinate chain contains a cyclodextrin moiety. Ifthe branching subordinate chain contains a cyclodextrin moiety, thecyclodextrin moiety may facilitate inclusion complex formation as wellas encapsulation of a therapeutic agent. Preferably, a cyclodextrinmoiety of a branching subordinate chain facilitates inclusion complexformation and encapsulation of a therapeutic agent in conjunction with acyclodextrin moiety in the polymer backbone. A branchedcyclodextrin-containing polymer may be prepared by any means known inthe art including, but not limited to, derivatization (e.g.substitution) of a polymer (e.g. linear or branched PEI) with acyclodextrin monomer precursor. Examples of polymers having prendantcyclodextrins are described in Tojima, et al., J. Polym. Sci. Part A:Polym. Chem. 36, 1965 (1998), Crini, et al., Eur. Polym. J. 33, 1143,(1997), Weickenmeier et al., Maromol. Rapid Commun. 17, 731 (1996), andBachmann, et al., J. Carbohydrate Chemistry 17, 1359 (1998); each ofwhich is incorporated here by reference. (The Weickenmeier articledescribes cyclodextrin sidechain polyesters, their synthesis andinclusion of adamantane derivatives.) The branchedcyclodextrin-containing polymer may be crosslinked as discussed above.

A poly(ethylenimine) (PEI) for use in the invention has a weight averagemolecular weight of between about 800 and about 800,000 daltons,preferably, between about 2,000 and 100,000 daltons, more preferably,between about 2,000 and about 25,000 daltons. The PEI may be linear orbranched. Suitable PEI compounds are commercially available from manysources, including pblyethylenimine from Aldrich Chemical Company,polyethylenimine from Polysciences, and POLYMIN poly(ethylenimine) andLUPASOL™ poly(ethylenimine) available from BASF Corporation.

Other Host-Functional Polymers

As discussed above, at least one polymer of the particulate composite isa polymer capable of forming an inclusion complex. Polymers havingpreferred cyclodxtrin host functionality, along with various methods ofpreparation, have been described above. In the same manner any polymer,linear or branched, having host functionality may be used in thepractice of this invention. Other examples of suitable “hosts” which maybe employed with the polymer include, but are not limited to, cavitands,crown ethers, cryptands, cucurbiturils, calixarenes, spherands, and thelike. Polymers of these other hosts may be prepared in the same way asdescribed above for the cyclodextrin-containing polymers. The host ofinterest may be derivatized through a functional group such as ahydroxyl group to attach a leaving group such as iodide, tosylate, etc.and reacted with a suitable comonomer A displacing the leaving group andforming the host copolymer. Alternatively, the host may contain or bederivatized to contain a functional group such as an amine or carboxylgroup allowing the host to undergo a condensation reaction with acomonomer A to form the host copolymer. Host copolymers, then, may beprepared having a mixture of host functionalities in the polymerbackbone as well as, if the copolymer is branched, in the branches.

Guest Functional Polymers

Guest functional polymers may be any polymer capable of forming aninclusion complex with a host-functional complexing agent. Typically theguest functionality will be present on a side chain or end-group. Anexample of a polymer having guest functionality not as part of thepolymer backbone would be a polymer having pendant adamantane groups.Examples of inclusion functionality which may be incorporated into thepolymer include those known in the art such as, but not limited to,adamantane, diadamantane, naphthalene, and cholesterol.

B. The Therapeutic Agent

According to the invention, at least one therapeutic agent becomesencapsulated in the polymer to form the particulate composite, asdescribed above. The term “therapeutic agent” is intended to encompassany active agent which has pharmacological or therapeutic use and, asdiscussed below, as active compounds or agents having microbidical uses.Examples of such therapeutic agents (or active agents) are discussedbelow. Encapsulation is defined as any means by which the therapeuticagent associates (e.g. electrostatic interaction, hydrophobicinteraction, actual encapsulation) with the polymer. The degree ofassociation may be determined by techniques known in the art including,for example, fluorescence studies, DNA mobility studies, lightscattering, electron microscopy, and will vary depending upon thetherapeutic agent. As a mode of delivery, for example, a therapeuticcomposition containing a multi-dimensional polymer network created fromthe polymer of a particulate composite, as described above, and DNA maybe used to aid in transfection, i.e. the uptake of DNA into an animal(e.g. human) cell. (Boussif, O. Proceedings of the National Academy ofSciences, 92:7297-7301 (1995); Zanta et al. Bioconjugate Chemistry,8:839-844 (1997); Gosselin et al. “Efficient Gene Transfer UsingReversibly Cross-Linked Low Molecular Weight Polyethylenimine”, Collegeof Pharmacy, The Ohio State University, published on web, revisedmanuscript Jul. 5, 2001.)). When the therapeutic agent is nucleicacid-based (e.g. DNA), the polymer the therapeutic agent forming thecomposite may be in the form of a “polyplex.” A polyplex is a compositebetween nucleic acids and accounting polymers. See, Feigner, et al.“Nomenclature for Synthetic Gene Delivery Systems”. Hum. Gene Ther. 8,511-512 (1997).

Any therapeutic agent mixture of therapeutic agents may be used with acomposition of the invention. Upon forming the particulate composite,the therapeutic agent may or may not retain its biological ortherapeutic activity. Upon release from the therapeutic composition,specifically, from the polymer of the particulate composite, theactivity of the therapeutic agent is restored. Or, in the case ofprodrug the potential for activity is restored. Accordingly, theparticulate composite advantageously affords the therapeutic agentprotection against loss of activity due to, for example, degradation andoffers enhanced bioavailability. Thus, a composition of the inventionmay be used to provide stability, particularly storage or solutionstability, to a therapeutic agent or any active chemical compound.Encapsulation of a lipophilic therapeutic agent offers enhanced, if notcomplete, solubility of the lipophilic therapeutic agent. Thetherapeutic agent may be further modified with a ligand prior to orafter particulate composite or therapeutic composition formation.

The therapeutic agent may be any lipophilic or hydrophilic, synthetic ornaturally occurring biologically active therapeutic agent includingthose known in the art. The Merck Index, An Encyclopedia of Chemicals,Drugs, and Biologicals, 13th Edition, 2001, Merck and Co., Inc.,Whitehouse Station, N.J. Examples of such therapeutic agents include,but are not limited to, small molecule pharmaceuticals, antibiotics,steroids, polynucleotides (e.g. genomic DNA, cDNA, mRNA, antisenseoligonucleotides, viruses, and chimeric polynucleotides), plasmids,peptides, peptide fragments, small molecules (e.g. doxorubicin),chelating agents (e.g. deferoxamine (DESFERAL),ethylenediaminetetraacetic acid (EDTA)), natural products (e.g. Taxol,Amphotericin), and other biologically active macromolecules such as, forexample, proteins and enzymes. See also U.S. Pat. No. 6,048,736 whichlists active agents (therapeutic agents) used as the guest to forminclusion compounds with cyclodextrin polymers. The disclosure of U.S.Pat. No. 6,048,736 is incorporated herein by reference. Small moleculetherapeutic agents may not only be the therapeutic agent within thecomposite particle but, in an additional embodiment, may be covalentlybound to a polymer in the composite. Preferably, the covalent bond isreversible (e.g. through a prodrug form or biodegradable linkage such asa disulfide) and provides another way of delivering the therapeuticagent.

2. The Complexing Agent

According to the invention, a complexing agent is a compound having hostor guest functionality that is capable of forming an inclusion complexwith a polymer in the particulate composite having the correspondingguest or host functionality. As described above, a guest complexingagent may be used to modify a polymer of the particulate compositehaving host functionality or a monomer of the polymer having hostfunctionality to form an inclusion complex. Also as described above, ahost complexing agent may form an inclusion complex with at least onepolymer of the particulate composite by acting as a host to the polymerguest functionality. The complexing agent may have two or more inclusionfunctionalities. For example, a complexing agent having two inclusionfunctionalities may be a guest, guest; a host, host; or a host, guestcomplexing agent. A complexing agent may also have a mixture of multiplehost and/or guest functionalities. The complexing agent also contains afunctional group which adds a beneficial property to the composition ofthe invention. This functional group may be, for example, a ligand, ahydrophilic or hydrophobic group, an additional therapeutic agent, etc.The complexing agent may also include a spacer group between theinclusion guest or host and the functional group.

Preferably, a complexing agent exhibits binding constants of about >10²,preferably, about >10³, and more preferably, about >10⁴. Typically,binding constants will range from about 10²-10⁶. Examples of inclusionguests suitable for the complexing agents include those known in the artsuch as, but not limited to, adamantane, diadamantane, naphthalene,cholesterol and derivatives thereof. Preferably, adamantane ordiadamantane is used. Amiel et al., Int. J. Polymer Analysis &Characterization, Vol. 1, 289-300 (1995); Amiel et al., Journal ofInclusion Phenomena and Molecular Recognition in Chemistry, 25:61-67(1996); Amiel et al., Advances in Colloid and Interface Science, 79,105-122 (1999); and Sandier et al., Langmuir, 16, 1634-1642 (2000).

A complexing agent contains a functional group that provides a benefitto the composition of the invention. A functional group may be as simpleadding a hydroxyl or amine functionality is one way to introducefunctionalty. In a preferred embodiment, the complexing agent may forman inclusion complex with a polymer of the particulate composite as wellas alter the composite, for example, to facilitate cell contact,intercellular trafficking, and/or cell entry and release. Any such groupknown in the art may be used. Examples of suitable “functional” groupsinclude, but are not limited, to ligands, nuclear localization signals(See Zanta et al., Proc. Natl. Acad. Sci. USA, 96, pp. 91-96 (1999),endosomal release peptides, endosomal release polymers, membranepermeabilization agents, or mixtures thereof. The nuclear localizationsignal (NLS) may be any nuclear localization signal known in the art.The endosomal release peptide or polymer may be any endosomal releasepeptide or polymer known in the art (e.g., HA-2 and GALA). See “Genedelivery by negatively charged ternary complexes of DNA, cationicliposomes and transferrin or fusigenic peptides” Simoes S, Slepushkin V,Gaspar R, de Lima MCP, Duzguries N, GENE THERAPY 5: (7) 955-964 July1998. An example of a cell membrane permeabilizing (or cell membranepermeable agent) is the TAT protein from HIV-1. The TAT protein is viraltranscriptional activator that is actively imported into the cellnucleus. Torchilin, V. P. et al, PNAS. 98, 8786-8791, (2001).

The complexing agent may also be functionalized with polymers thatincrease solubility and/or impart stabilization, particularly underbiological conditions. Stabilization of the composition may be achievedor enhanced by the use of complexing agents having hydrophillic groupsor lipophillic groups. A preferred type of hydrophilic group ispolyethlene glycol or a polyethylene glycol-containing copolymer (PEG).Preferred polyethylene ethylene glycols have the formulaHO(CH₂CH₂O)_(z)H, where z varies from 2 to 500, preferably 10-300. PEG600, PEG 3400, and PEG 5000 are representative of the polyethyleneglycols which may be used in the invention. In general, the higher themolecular weight of the PEG in the complexing agent the greater of thestabilization of the composition. Higher molecular weight PEG's aregenerally preferred. A preferred complexing agent is pegylatedadamantane or pegylated diadamantane. The structures of someAdamantane-PEG molecules useful as complexing agents are shown inFIG. 1. To increase lipophilicity (hydrophobicity), the complexing agentmay contain lipophilic groups such as long chain alkyls, fatty acids,etc. Choice of the lipophilic group depends on the amount oflipophilicity desired. As can be seen from this discussion, thecomplexing agent may be modified with any type of functionality tointroduce a desired property into the composition. The complexing agentmay be prepared using standard organic techniques. Employing mixtures ofdifferent complexing agents allows for greater variation and specificityin achieving desired composition properties.

A spacer group may be used to join the functional group to thecomplexing agent. The spacer group may be any spacer group known in theart which does not adversely effect the properties of the guestcomplexing agent or the functional group. For example, the spacer groupmay be a direct link, such that the functional group is bound directlyto the complexing agent. Alternatively, the spacer group may be a moietythat is water soluble, highly anionic at physiological pH or hasfusogenic abilities under acidic conditions. Preferably, the spacergroup enhances the binding affinity of the complexing agent with thepolymer in the inclusion complex (e.g., an anionic spacer groupcontaining glutamic acid residues, carboxylic acid groups, etc.). Thespacer group may also contain a reducible link (e.g., disulfide linkage)reduction of which would release the functional group from thecomplexing agent. Examples of suitable spacer groups include, but arenot limited to, a direct link, polyglutamic acid, GALA, and polyethyleneglycols (PEG).

The functional group may also be an additional therapeutic agent. Thetherapeutic agent may be reversibly bound to the complexing agent (e.g.through a prodrug form or biodegradable linkages). This provides a wayof delivering additional therapeutic agents via the complexing agent.

A preferred class of complexing agents having adamantane guestfunctionality are compounds of the formula:

wherein

-   J is —NH—, —(C═O)NH—(CH₂)_(d)—, —NH—C(═O)—(CH₂)—CH₂SS—,    —C(═O)O——(CH₂), O—P(═O)(O—(CH₂)_(e)-Ad)O—,

a peptide or polypeptide residue, or

—NH—(C═O)—CH(R¹)—NH—(C═O)—CH(R²)—NH—;

-   Ad is adamantyl;-   R¹ is —(CH₂)_(a)—C0₂H, an ester or salt thereof; or    —(CH₂)_(a)—CONH₂;-   PEG is —O(CH₂CH₂O)₂—, where z varies from 2 to 500;-   L is H, —NH₂, —NH—(C═O)—(CH₂)_(e)—(C═O)—CH₂—, —S(═O)₂—HC═CH₂—, —SS—,    —C(═O)O— or a carbohydrate residue;-   a is 0 or 1;-   b is 0 or 1;-   d ranges from 0 to 6;-   e ranges from 1 to 6;-   y is 0 or 1; and-   x is 0 or 1.

By use of a functionalized complexing agents, a therapeutic compositionof the invention may be modified or functionalized to facilitate cellcontact and/or cell entry. To achieve multiple functions and/orbenefits, the composition may form two or more types of inclusioncomplexes using complexing agents having different functionalities. Asdescribed above, a ligand may be used to modify a polymer of theparticulate composite or a complexing agent. Thus, according to theinvention, a composition of the invention may, via the inclusioncomplex, contain more than one ligand and thus bear more than one sitefor cell targeting and/or delivery. The particulate composite havingmultiple ligand- or other-functionalized complexing agents may bestabilized by adding complexing agents with stabilization or solubilityfunctionality such as the pegylated complexing agents.

Because the polymer may form multiple inclusion complexes with a mixtureof different functionalized complexing agents, a therapeutic compositionof the invention may contain, for example, multiple therapeutic agents,different ligands and/or various stabilization polymers. Where thecomplexing agent is functionalized with therapeutic agent or a prodrug,forming multiple inclusion complexes allows for multiple therapeutics tobe delivered using the same therapeutic composition. If a ligand ispresent, the entire combination (or cocktail) of therapeutic agents maybe directed to a specific cell type, disease, or other therapeutic use.

A functionalized guest complexing agent may be prepared by any meansknown in the art. See Amiel et al., Int. J. Polymer Analysis &Characterization, Vol. 1, 289-300 (1995); Amiel et al., Journal ofInclusion Phenomena and Molecular Recognition in Chemistry, 25, 61-67(1996); Sandier et al., Langmuir, 16, 1634-1642 (2000).

3. Preparation of a Composition of the Invention

The invention also relates to method of preparing a composition. Themethod combines a therapeutic agent, a polymer having host or guestfunctionality, and a complexing agent to form the therapeuticcomposition. The complexing agent, acting as a guest or a host, forms aninclusion complex with the polymer. In another embodiment, the polymerand the therapeutic agent are first combined to form a particulatecomposite. The particulate composite is then combined with thecomplexing agent to form an inclusion complex of the therapeuticcomposition. The composition may also be formed by first mixing thepolymer with the complexing agent and then combining that mixture withthe therapeutic agent to form the composite and, accordingly, acomposition of the invention.

A. Formation of the Polymer-Agent Particulate Composite

The particulate composite of a therapeutic agent and a polymer may beprepared by any suitable means known in the art. For example, aparticulate composite may be formed by simply contacting, mixing, ordispersing a therapeutic agent with a polymer. For example, the polymerand the therapeutic agent may be mixed in a solvent in which both aresoluble, in which the polymer is soluble but the therapeutic agent isdispersed, or in a solvent which disperses the polymer and thetherapeutic agent but solubilizes the particulate composite. Forpharmaceutical applications, the solvent may be any physiologicallyacceptable aqueous solution. The particulate composite may be formed bythe association of the polymer and the therapeutic agent, selfassociation of the polymer, or by chemical means. Prior to formation ofthe particulate composite, the polymer of the particulate compositegenerally does not exist as a substantially associated structure suchas, for example, a polymer gel. However, the polymer as part of theparticulate composite, depending upon the nature of the polymers and thetherapeutic agent, may form a substantially associated structure such asa gel. A particulate composite may also be prepared by polymerizingmonomers, which may be the same or different, to form a linear orbranched polymer in the presence of a therapeutic agent. A particulatecomposite may also be prepared by polymerizing monomers, which may bethe same or different, capable of forming a linear or branched polymerin the presence of a therapeutic agent where the therapeutic agent actsas a template for the polymerization. Trubetskoy et al., Nucleic AcidsResearch, Vol. 26, No. 18, pp. 4178-4185 (1998).

The amount of polymer and therapeutic agent employed may be any amountwhich allows the particulate composite to assemble. Typically thepolymer will be used in excess of the therapeutic agent. When thepolymer used to form the polymer carries a cationic or anionic charge,such as with a cationically charged comonomer A or with a polyalkyleneimine such as PEI and when the therapeutic agent carries a charge suchas an anionic polynucleotide, the ratio of polymer to therapeutic agentmay be expressed as a charge ratio. The charge ratio is an expression ofthe ratio of charge of the polymer to that of the therapeutic agent. Asshow in the examples particulate composites of cationic cyclodextrinpolymers and anionic DNA are typically formulated at 5+/− charge ratio,that is five cationic charges from the cyclodextrin polymer to oneanionic charge of DNA. The charge ratio may be any ratio that allows theparticulate composite to form and may be in excess of the minimum chargeratio necessary. Where the polymer and/or the therapeutic agent isuncharged, the amount or ratio of the polymer to therapeutic agent maybe expressed in terms of weight, moles or concentration as in known inthe art.

According to the invention, the polymer of the particulate composite mayalso be treated under conditions sufficient to form a particulatecomposite comprising a therapeutic agent and a multi-dimensional polymernetwork. Such multi-dimensional polymer networks are described in WO00/33885, which is incorporated here by reference. As described in WO00/33885, treating of the polymer of the particulate composite underconditions sufficient to form a multi-dimensional polymer network may beaccomplished using any suitable reaction condition(s), including theaddition of additional agents or reactants, that promote association ofthe polymer and the therapeutic agent of the particulate composite. Thepolymer may be associated via interpolymer covalent bonds, noncovalentbonds (e.g. ionic bonds), or noncovalent interactions (e.g. van derWaals interactions). Association via intrapolymer covalent bonding,noncovalent bonding, or noncovalent interactions of the polymer mayoccur as well. As a result of such association, the polymer of theparticulate composite interacts to form a multi-dimensional polymernetwork.

In one embodiment of the invention, to form a particulate compositecomprising a therapeutic agent and a multi-dimensional polymer networkinvolves crosslinking reactions. For example, if the polymer of theparticulate composite is a single polymer molecule, the polymer may bereacted with a molecule(s), oligomer(s), or different polymer(s) thatpromotes crosslinking or forms crosslinks such that intrapolymercrosslinking of or actual crosslinking with the single polymer moleculeof the particulate composite results. Similarly, if the polymer of theparticulate composite is a mixture of two or more polymers, the polymeror polymers may be reacted with a molecule(s), oligomer(s), or differentpolymer(s) that promotes crosslinking or forms crosslinks. The resultingcrosslinking may be intrapolymer and/or interpolymer, preferablyinterpolymer, crosslinking of the polymer or polymers of the particulatecomposite.

The crosslinking agent may be any crosslinking agent known in the art.The crosslinking agent may be any oligomer or polymer (e.g. polyethyleneglycol (PEG) polymer, polyethylene polymer) capable of promotingcrosslinking within or may be actually crosslinking with the polymer ofthe particulate composite. The crosslinking oligomer or polymer may bethe same or different as the polymer of the particulate composite.Likewise, the crosslinking agent may be any suitable molecule capable ofcrosslinking with the polymer of the particulate composite. Thecrosslinking agent may itself contain a ligand.

The degree of association, as described in WO 00/33885, of the polymerof the particulate composite forming the multi-dimensional polymernetwork may vary from partial association to complete association. Byvarying the degree of association of the polymer, a short chain polymermay be made to exhibit the characteristics of a long chain polymer whileretaining the desired characteristics of a short chain polymer upondisassociation. For example, long chain polymer character promotesoverall stability, i.e. resistance to degradation, until the target cellis reached while short chain polymer character promotes DNA releasewithin the target cell. This duality affords a therapeutic compositioncontaining a therapeutic agent and a multi-dimensional polymer networkthat exhibits improved stability in both nonphysiological andphysiological conditions and greater shelf-life stability. Varying thedegree of association of the polymer of the therapeutic composition alsopermits controlled release of the therapeutic agent.

The particle size of the particulate composite depends upon the polymerand therapeutic agent used to form the composition of the invention. Asshown in the examples which follow, particulate sizes may range from50-1000 nm, preferably 50-500 nm. Forming the inclusion complextypically does not significantly increase particle size. Thecompositions remain as discreet particles. As discussed below,compositions containing pegylated complexing agents show excellentstability in salt solutions. Advantageously, the compositions are stableat physiological conditions allowing their use as delivery vehicles fortherapeutic agents and in the treatment of various diseases anddisorders.

B. Formation of the Inclusion Complex

The inclusion complex may be prepared by any suitable means known in theart. For example, the inclusion complex may be formed by simplycontacting, mixing, or dispersing the particulate composite and thecomplexing agent. For example, the particulate composite and thecomplexing agent may be mixed in a solvent in which both are soluble, inwhich the particulate composite or the complexing agent is soluble butthe other is dispersed, or in a solvent which disperses the particulatecomposite and the complexing agent but solubilizes the inclusioncomplex. Preferably, the inclusion complex is formed by adding thecomplexing agent to the particulate composite in the same vessel as usedto mix the polymer and the therapeutic agent to form the inclusioncomplex. For pharmaceutical applications, the solvent may be anyphysiologically acceptable aqueous solution.

The complexing agent may be added to the composite particle in any molarratio to the moles of host and/or guest functionality present in thepolymer of the composite which forms the inclusion complex. In general,the complexing agent is added in a 1:1 molar ratio to the moles hostand/or guest functionality. Lower molar ratios (excess host and/or guestfunctionality on the polymer) may be used as long as the compositioncontains at least one complexing agent and at least one host or guestfunctionality on the polymer to form an inclusion complex. Excesscomplexing agent may also be used. Typically, then, the molar ratio ofcomplexing agent to moles of polymer host and/or guest functionalityranges from 0.01:1 to 1:0.01, and preferably is between 0.5:1 and 1:0.5.When multiple complexing agents are used, the molar ratio of theindividual complexing agents may be chosen by the desired functionalityto be introduced into the composition. For example, it may be in a givencomposition that a pegylated stabilizing complexing agent is present ina 0.9:1 molar ratio and a complexing agent containing a ligand may bepresent in only minor amounts, e.g., 1-2% of the complexing agent. Thetotal amount complexing agent in such a composition typically fallswithin the ranges discussed above.

4. Compositions and Methods of Treatment

A therapeutic composition of the invention may be formulated as a solid,liquid, suspension, or emulsion. Preferably a therapeutic composition ofthe invention is in a form that can be injected intravenously. Othermodes of administration of a therapeutic composition of the inventioninclude methods known in the art such as, but not limited to, oraladministration, inhalation, topical application, parenteral,intravenous, intranasal, intraocular, intracranial or intraperitonealinjection, and pulmonary administration. The method of administrationoften depends on the formulation of the therapeutic composition. Priorto administration, a therapeutic composition may be isolated andpurified by any means known in the art including, for example,centrifugation, dialysis and/or lyophilization.

The invention relates to pharmaceutical compositions which comprise aneffective amount of a therapeutic composition of the invention and apharmaceutically and physiologically acceptable carrier. Suitable solidor liquid galenic formulations are, for example, granules, powders,coated tablets, microcapsules, suppositories, syrups, elixirs,suspensions, emulsions, drops or injectable solutions. Commonly usedadditives in pharmaceutical compositions include, but are not limitedto, preparations are excipients, disintegrates, binders, coating agents,swelling agents, glidants, or lubricants, flavors, sweeteners orsolubilizers. More specifically, frequently used additives are, forexample, magnesium carbonate, titanium dioxide, lactose, mannitol andother sugars, talc, lactalbumin, gelatin, starch, cellulose and itsderivatives, animal and vegetable oils, polyethylene glycols andsolvents. The solvents include sterile water and monohydric orpolyhydric alcohols such as glycerol.

Depending upon the type of therapeutic agent used, a therapeuticcomposition of the invention may be used in a variety of therapeuticmethods (e.g. DNA vaccines, antibiotics, antiviral agents) for thetreatment of inherited or acquired disorders such as, for example,cystic fibrosis, Gaucher's disease, muscular dystrophy, AIDS, cancers(e.g., multiple myeloma, leukemia, melanoma, and ovarian carcinoma),cardiovascular conditions (e.g., progressive heart failure, restenosis,and hemophilia), and neurological conditions (e.g., brain trauma).According to the invention, a method of treatment administers to aperson or mammal in recognized need of the therapeutic a therapeuticallyeffective amount of a therapeutic composition of the invention. Atherapeutically effective amount, as recognized by those of skill in theart, will be determined on a case by case basis. Factors to beconsidered include, but are not limited to, the disorder to be treatedand the physical characteristics of the one suffering from the disorder.

6. Other Utilities

The inclusion complexes of the invention may also find utility indelivering chemicals used in the agricultural industry. In anotherembodiment of the invention, the “therapeutic agent” is a biologicallyactive compound having microbicidal and agricultural utility. Thesebiologically active compounds include those known in the art. Forexample, suitable agriculturally biologically active compounds include,but are not limited to, fertilizers, fungicides, herbicides,insecticides, and mildewcides. Microbicides are also used inwater-treatment to treat muncipal water supplies and industrial watersystems such as cooling waters, white water systems in papermaking.Aqueous systems susceptible to microbiological attack or degradation arealso found in the leather industry, the textile industry, and thecoating or paint industry. Examples of such microbicides and their usesare described, individually and in combinations, in U.S. Pat. Nos.5,693,631, 6,034,081, and 6,060,466, which are incorporated herein byreference, compositions containing active agents such as those discussedabove may be used in the same manner as known for the active ingredientitself. Notably, because such uses are not pharmacological uses, thepolymer of the composite does not necessarily have to meet the toxicityprofile required in pharmaceutical uses.

7. Examples

The following examples are given to illustrate the invention. It shouldbe understood, however, that the invention is not to be limited to thespecific conditions or details described in these examples.

Materials. β-cyclodextrin (Cerestar USA, Inc. of Hammond, Ind.) wasdried in vacuo (<0.1 mTorr) at 120° C. for 12 h before use.Biphenyl-4,4′-disulfonyl chloride (Aldrich Chemical Company, Inc. ofMilwaukee, Wis.) was recrystallized from chloroform/hexanes. Potassiumiodide was powdered with a mortar and pestle and dried in an oven at200° C. All other reagents were obtained from commercial suppliers andwere used as received without further purification. Polymer samples wereanalyzed on a Hitachi HPLC system equipped with an Anspec RI detector, aPrecision Detectors DLS detector, and a Progel-TSK G3000_(PWXL) columnusing 0.3 M NaCl or water as eluant at a 1.0 mL min⁻¹ flow rate.

Example 1 Biphenyl-4,4′-disulfonyl-A, D-Capped β-Cyclodextrin, 1(Tabushi et al. J. Am. Chem. Soc. 106, 5267-5270 (1984))

A 500 mL round bottom flask equipped with a magnetic stirbar, a Schlenkadapter and a septum was charged with 7.92 g (6.98 mmol) of dryβ-cyclodextrin and 250 mL of anhydrous pyridine (Aldrich ChemicalCompany, Inc.). The resulting solution was stirred at 50° C. undernitrogen while 2.204 g (6.28 mmol) of biphenyl-4, 4′-disulfonyl chloridewas added in four equal portions at 15 min intervals. After stirring at50° C. for an additional 3 h, the solvent was removed in vacuo and theresidue was subjected to reversed-phase column chromatography using agradient elution of 0-40% acetonitrile in water. Fractions were analyzedby high performance liquid chromatography (HPLC) and the appropriatefractions were combined. After removing the bulk of the acetonitrile ona rotary evaporator, the resulting aqueous suspension was lyophilized todryness. This afforded 3.39 g (38%) of 1 as a colorless solid.

Example 2 6^(A), 6^(D)-Diiodo-6^(A), 6^(D)-Dideoxy-β-cyclodextrin, 2(Tabushi et al. J. Am. Chem. 106, 4580-4584 (1984))

A 40 mL centrifuge tube equipped with a magnetic stirbar, a Schlenkadapter and a septum was charged with 1.02 g (7.2 mmol) of 1, 3.54 g(21.3 mmol) of dry, powdered potassium iodide (Aldrich) and 15 mL ofanhydrous N,N-dimethylformamide (DMF) (Aldrich). The resultingsuspension was stirred at 80° C. under nitrogen for 2 h. After coolingto room temperature, the solids were separated by filtration and thesupernatant was collected. The solid precipitate was washed with asecond portion of anhydrous DMF and the supernatants were combined andconcentrated in vacuo. The residue was then dissolved in 14 mL of waterand cooled in an ice bath before 0.75 mL (7.3 mmol) oftetrachloroethylene (Aldrich) was added with rapid stirring. Theprecipitated product was filtered on a medium glass frit and washed witha small portion of acetone before it was dried under vacuum over P₂O₅for 14 h. This afforded 0.90 g (92%) of 2 as a white solid.

Example 3 6^(A), 6^(D)-Diazido-6^(A), 6^(D)-Dideoxy-β-cyclodextrin, 3(Tabushi et al. Tetrahedron Lett. 18, 1527-1530 (1977))

A 100 mL round bottom flask equipped with a magnetic stirbar, a Schlenkadapter and a septum was charged with 1.704 g (1.25 mmol) ofβ-cyclodextrin diiodide, 0.49 g (7.53 mmol) of sodium azide (EM Scienceof Gibbstown, N.J.) and 10 mL of anhydrous N,N-dimethylformamide (DMF).The resulting suspension was stirred at 60° C. under nitrogen for 14 h.The solvent was then removed in vacuo. The resulting residue wasdissolved in enough water to make a 0.2 M solution in salt and thenpassed through 11.3 g of Biorad AG501-X8(D) resin to remove residualsalts. The eluant was then lyophilized to dryness yielding 1.232 g (83%)of 3 as a white amorphous solid which was carried on to the next stepwithout further purification.

Example 4 6^(A), 6^(D)-Diamino-6^(A), 6^(D)-Dideoxy-β-cyclodextrin, 4(Mungall et al., J. Org. Chem. 1659-1662 (1975))

A 250 mL round bottom flask equipped with a magnetic stirbar and aseptum was charged with 1.232 g (1.04 mmol) of β-cyclodextrin bisazideand 50 mL of anhydrous pyridine (Aldrich). To this stirring suspensionwas added 0.898 g (3.42 mmol) of triphenylphosphine. The resultingsuspension was stirred for 1 h at ambient temperature before 10 mL ofconcentrated aqueous ammonia was added. The addition of ammonia wasaccompanied by a rapid gas evolution and the solution becamehomogeneous. After 14 h, the solvent was removed in vacuo and theresidue was triturated with 50 mL of water. The solids were filtered offand the filtrate was made acidic (pH<4) with 10% HCl before it wasapplied to an ion exchange column containing Toyopearl SP-650M (NH₄ ⁺form) resin. The product 4 was eluted with a gradient of 0-0.5 Mammonium bicarbonate. Appropriate fractions were combined andlyophilized to yield 0.832 g (71%) of the product 4 as the bis(hydrogencarbonate) salt.

Example 5 β-cyclodextrin-DSP copolymer, 5

A 20 mL scintillation vial was charged with a solution of 92.6 mg(7.65×10⁻⁵ mol) of the bis(hydrogen carbonate) salt of 4 in 1 mL ofwater. The pH of the solution was adjusted to 10 with 1 M NaOH before asolution of 30.9 mg (7.65×10⁻⁵ mol) of dithiobis(succinimidylpropionate) (DSP, Pierce Chemical Co. of Rockford, Ill.) in 1 mL ofchloroform was added. The resulting biphasic mixture was agitated with aVortex mixer for 0.5 h. The aqueous layer was then decanted andextracted with 3×1 mL of fresh chloroform. The aqueous polymer solutionwas then subjected to gel permeation chromatography (GPC) on ToyopearlHW-40F resin using water as eluant. Fractions were analyzed by GPC andappropriate fractions were lyophilized to yield 85 mg (85%) as acolorless amorphous powder.

Example 6 β-cyclodextrin-DSS copolymer, 6

A β-cyclodextrin-DSS copolymer, 6, was synthesized in a manner analogousto the DSP polymer, 5, except that disuccinimidyl suberate (DSS, PierceChemical Co. of Rockford, Ill.) was substituted for the DSP reagent.Compound 6 was obtained in 67% yield.

Example 7 β-cyclodextrin-DTBP copolymer, 7

A 20 mL scintillation vial was charged with a solution of 91.2 mg(7.26×10⁻⁵ mol) of the bis(hydrogen carbonate) salt of 4 in 1 mL ofwater. The pH of the solution was adjusted to 10 with 1 M NaOH before22.4 mg (7.26×10⁻⁵ mol) of dimethyl 3,3′-dithiobis(propionimidate).2 HCl(DTBP, Pierce Chemical Co. of Rockford, Ill.) was added. The resultinghomogeneous solution was agitated with a Vortex mixer for 0.5 h. Theaqueous polymer solution was then subjected to gel permeationchromatography (GPC) on Toyopearl HW-40F resin. Fractions were analyzedby GPC and appropriate fractions were lyophilized to yield 67 mg (67%)of a colorless amorphous powder.

Example 8 Polyethylene glycol (PEG) 600 diacid chloride, 8

A 50 mL round bottom flask equipped with a magnetic stirbar and a refluxcondenser was charged with 5.07 g (ca. 8.4 mmol) of polyethylene glycol600 diacid (Fluka Chemical Corp of Milwaukee, Wis.) and 10 mL ofanhydrous chloroform (Aldrich). To this stirring solution was added 3.9mL (53.4 mmol) of thionyl chloride (Aldrich) and the resulting solutionwas heated to reflux for 1 h, during which time gas evolution wasevident. The resulting solution was allowed to cool to room temperaturebefore the solvent and excess thionyl chloride were removed in vacuo.The resulting oil was stored in a dry box and used without purification.

Example 9 β-cyclodextrin-PEG 600 copolymer, 9

A 20 mL scintillation vial was charged with a solution of 112.5 mg(8.95×10⁻⁵ mol) of the bis(hydrogen carbonate) salt of 6^(A),6^(D)-diamino-6^(A), 6^(D)-dideoxy-β-cyclodextrin(4), 50 μL (3.6×10⁻⁴mol) of triethylamine (Aldrich), and 5 mL of anhydrousN,N-dimethylacetamide (DMAc, Aldrich). The resulting suspension was thentreated with 58 mg (9.1×10⁻⁵ mol) of polyethylene glycol 600 diacidchloride, 8. The resulting solution was agitated with a Vortex mixer for5 minutes and then allowed to stand at 25° C. for 1 h during which timeit became homogeneous. The solvent was removed in vacuo and the residuewas subjected to gel permeation chromatography on Toyopearl HW-40F resinusing water as eluant. Fractions were analyzed by GPC and appropriatefractions were lyophilized to dryness to yield 115 mg (75%) of acolorless amorphous powder.

Example 10 6^(A), 6^(D)-Bis-(2-aminoethylthio)-6^(A),6^(D)-dideoxy-β-cyclodextrin, 10 (Tabuchi, I: Shimokawa, K; Fugita, K.Tetrahedron Lett. 1977, 1527-1530)

A 25 mL Schlenk flask equipped with a magnetic stirbar and a septum wascharged with 0.91 mL (7.37 mmol) of a 0.81 M solution of sodium2-aminoethylthiolate in ethanol. (Fieser, L. F.; Fiester, M. Reagentsfor Organic Synthesis; Wiley: New York, 1967; Vol. 3, pp. 265-266). Thesolution was evaporated to dryness and the solid was redissolved in 5 mLof anhydrous DMF (Aldrich). 6^(A), 6^(D)-Diiodo-6^(A),6^(D)-dideoxy-β-cyclodextrin (2) (100 mg, 7.38×10⁻⁵ mol) was added andthe resulting suspension was stirred at 60° C. under nitrogen for 2 h.After cooling to room temperature, the solution was concentrated invacuo and the residue was redissolved in water. After acidifying with0.1 N HCl, the solution was applied to a Toyopearl SP-650M ion-exchangecolumn (NH₄ ⁺ form) and the product was eluted with a 0 to 0.4 Mammonium bicarbonate gradient. Appropriate fractions were combined andlyophilized to dryness. This afforded 80 mg (79%) of 10 as a whitepowder.

Alternative Synthesis of dicysteamine β-CD 10.

To a solution of 4.69 g (3.17 mmol) of 2 in 100 mL of degassed water wasadded 0.489 g (6.34 mmol) of freshly sublimed cysteamine. The solutionwas stirred under reflux for 2 h. After cooling to room temperature andacidifying with 1N HCl, the solution was applied to a Toyopearl SP-650Mion-exchange column (NH₄ ⁺ form) and the product was eluted with a 0 to0.2M ammonium bicarbonate gradient. Appropriate fractions were combinedand lyophilized to dryness. This procedure gave 1.87 g (39% yield) of awhite solid. The solid was characterized by TLC (silica gel,n-PrOH—AcOEt—H₂O—NH₃aq 5/3/3/1, detection by ninhydrin) and exhibited amajor spot corresponding to 10. Matrix-assisted laserdesorption/ionization (MALDI) time-of flight (TOF) mass spectrum wasrecorded on 2 meter ELITE instrument supplied by PerSeptive Biosystems,Inc. MALDI-TOF m/z calcd for 3: 1252. found: 1253.5 [M+H]⁺, 1275.5[M+Na]⁺, 1291.4 [M+K]⁺. ¹³C NMR (Bruker 500 MHz, D₂O)* ppm: 32.1 (S—CH₂)and 38.8 (CH₂—NH₂), 32.9 (C6 adjacent to S), 60.2 (C6 adjacent to OH),70.8, 71.4, 72.5 (C2, C3, C5), 81.8 (C4), 101.7 (C1).

Example 11 β-cyclodextrin(cystamine)-DTBP copolymer, 11

A 4 mL vial was charged with a solution of 19.6 mg (1.42×10⁻⁵ mol) ofthe bis(hydrogen carbonate) salt of 10 in 0.5 mL of 0.1 M NaHCO₃. Thesolution was cooled in an ice bath before 4.4 mg (1.4×10⁻⁵ mol) ofdimethyl 3, 3′-dithiobispropionimidate-2 HCl (DTBP, Pierce Chemical Co.of Rockford, lllinois) was added. The resulting solution was thenagitated with a Vortex mixer and allowed to stand at 0° C. for 1 h. Thereaction was quenched with 1M Tris-HCl before it was acidified to pH 4with 0.1N HCl. The aqueous polymer solution was then subjected to gelpermeation chromatography on Toyopearl HW-40F resin. Fractions wereanalyzed by GPC and appropriate fractions were lyophilized to dryness.This afforded 21.3 mg (100%) of 11 as a white powder.

Example 12 β-Cyclodextrin(cystamine)-DMS copolymer, 12

A 10 mL Schlenk flask equipped with a magnetic stirbar and a septum wascharged with 200 mg (1.60×10⁻⁴ mol) of 10, 44 μL (3.2×10⁻⁴ mol) oftriethylamine (Aldrich Chemical Co., Milwaukee, Wis.), 43.6 mg(1.60×mol) of dimethylsuberimidate.2HCl (DMS, Pierce Chemical Co. ofRockford, Ill.), and 3 mL of anhydrous DMF (Aldrich Chemical Co.,Milwaukee, Wis.). The resulting slurry was heated to 80° C. for 18 hoursunder a steady stream of nitrogen during which time most of the solventhad evaporated. The residue which remained was redissolved in 10 mL ofwater and the resulting solution was then acidified with 10% HCl to pH4. This solution was then passed through an Amicon Centricon Plus-205,000 NMWL centrifugal filter. After washing with 2×10 mL portions ofwater, the polymer solution was lyophilized to dryness yielding 41.4 mg(18%) of an off-white amorphous solid.

Alternative Synthesis

β-Cyclodextrin(cystamine)-DMS copolymer was synthesized as describedpreviously (Gonzalez, et al. 1999). In a typical experiment, a 25 mLvial was charged with a solution of the bis(hydrogen carbonate) salt ofdicysteamine β-CD 10 (399.6 mg, 0.269 mmol) dissolved in 500 μL of 0.5MNa₂CO₃. Dimethylsuberimidate.2HCl (DMS, Pierce Chemical Co. of RockfordIll., 73.5 mg, 0.269 mmol) was added and the solution was centrifugedbriefly to dissolve the components. The resulting mixture was stirred at25° C. for 15 h. The mixture was then diluted with 10 mL of water andthe pH brought below 4 with the addition of 1N HCl. This solution wasthen dialyzed against a Spectra/Por 7 MWCO 3500 dialysis membrane(Spectrum) in dH₂O for 24 h. The dialyzed solution was lyophilized todryness. ¹³C NMR (Bruker 500 MHz, D₂O)*ppm: 25.8, 26.0, 27.0, 28.7,29.9, 32.2, 37.5, 38.1, 41.1, 60.0, 71.6, 72.3, 72.6, 80.8, 101.4,167.9.

Example 13 Fixed Permanent Charged Copolymer Complexation with Plasmid

In general, equal volumes of fixed charged CD-polymer and DNA plasmidsolutions in water are mixed at appropriate polymer/plasmid chargeratios. The mixture is then allowed to equilibrate and self-assemble atroom temperature. Complexation success is monitored by transferring asmall aliquot of the mixture to 0.6% agarose gel and checking for DNAmobility. Free DNA travels under an applied voltage, whereas complexedDNA is retarded at the well.

1 μg of DNA at a concentration of 0.1 μg/μL in distilled water was mixedwith 10 μL of copolymer 12 at polymer amine: DNA phosphate charge ratiosof 2.4, 6, 12, 24, 36, 60, and 120. 1 μg/μL of loading buffer (40%sucrose, 0.25% bromophenol blue, and 200 mM Tris-Acetate buffercontaining 5 mM EDTA (Gao et al., Biochemistry 35:1027-1036 (1996)) wasadded to each solution. Each DNA/polymer sample was loaded on a 0.6%agarose electrophoresis gel containing 6 μg of EtBr/100 mL in 1×TAEbuffer (40 mM Tris-acetate/1 mM EDTA) and 40V was applied to the gel for1 hour. The extent of DNA/polymer complexation was indicated by DNAretardation in the gel migration pattern. The copolymer (12) retardedDNA at charge ratios of 2 and above, indicating complexation under theseconditions.

Example 14 Transfection Studies with Plasmids Encoding LuciferaseReporter Gene

BHK-21 cells were plated in 24 well plates at a cell density of 60,000cells/well 24 hours before transfection. Plasmids encoding theluciferase gene were mixed with the CD-polymer as in Example 13. Mediasolution containing the DNA/polymer complexes was added to culturedcells and replaced with fresh media after 24 hours of incubation at 37°C. The cells were lysed 48 hours after transfection. Appropriatesubstrates for the luciferase light assay were added to the cell lysate.Luciferase activity, measured in terms of light units produced, wasquantified by a luminometer. DNA/polymer complexes successfullytransfected BHK-21 cells at a charge ratios above 3 with maximumtransfection at polymer amine:DNA phosphate charge ratio of 40. Celllysate was also used to determine cell viability by the Lowry proteinassay. (Lowry et al., Journal of Biological Chemistry, Vol. 193, 265-275(1951)). No toxicity was observed up to charge ratios of 40.

Example 15 Synthesis of β-cyclodextrin(cystamine)-DMA copolymer, 13

A 20 mL scintillation vial equipped with a magnetic stirbar was chargedwith 180 mg (0.131 mmol) of 10 and 32 mg of dimethyl adipimidate (DMA,Pierce Chemical Co. of Rockford, Blinois). To this was added 500 μL of0.5 M Na₂CO₃. The resulting solution was covered with foil and stirredovernight. The mixture was acidified with 0.1 N HCl and dialyzed withSpectrapor MWCO 3,500 membrane for 2 days and lyophilized to afford 41mg of a white amorphous solid with Mw=6 kDa, as determined by lightscattering.

Example 16 Synthesis of β-cyclodextrin(cystamine)-DMP copolymer, 14

A 20 mL scintillation vial equipped with a magnetic stirbar was chargedwith 160 mg (0.116 mmol) of 10 and 30.1 mg of dimethyl pimelimidate(DMP, Pierce Chemical Co. of Rockford, Ill.). To this was added 500 μLof 0.5 M Na₂CO₃. The resulting solution was covered with foil andstirred overnight. The mixture was then acidified with 0.1 N HCl anddialyzed with Spectrapor MWCO 3,500 membrane for 2 days and lyophilizedto afford 22 mg of a white amorphous solid with Mw=6 kDa, as determinedby light scattering.

Example 17 β-cyclodextrin(cystamine)-PEG600 Copolymer, 15

A 100 mL round bottom flask equipped with a magnetic stirbar, a Schlenkadapter and a septum was charged with 1.564 g (1.25 mmol) of 10 and 25mL of freshly distilled dimethylacetamide (DMAc, Aldrich). To the slurrywas added 0.7 mL (4 eq) of triethylamine and a solution of 8 (2.39 g,3.75 eq) in 5 mL of DMAc. The resulting solution was agitated withVortex mixer for 5 minutes and then allowed to stand at 25° C. for 1hour during which time it became homogeneous. The solvent was removedunder vacuum and the residue was subjected to gel permeationchromatography on Toyopearl HW-40F resin using water as eluent.Fractions were analyzed by GPC and appropriate fractions werelyophilized to dryness to yield a colorless amorphous powder.

Example 18 Synthesis of β-cyclodextrin-Tosylate, 16 (Melton, L. D., andSlessor, K. N., Carbohydrate Research, 18, p. 29 (1971))

A 500 mL round-bottom flask equipped with a magnetic stirbar, a vacuumadapter and a septum was charged with a solution of dry β-cyclodextrin(8.530 g, 7.51 mmol) and 200 mL of dry pyridine. The solution was cooledto 0° C. before 1.29 g (6.76 mmol) of tosyl chloride was added. Theresulting solution was allowed to warm to room temperature overnight.The pyridine was removed as much as possible in vacuo. The resultingresidue was then recrystallized twice from 40 mL of hot water to yield7.54 (88%) of a white crystalline solid.

Example 19 Synthesis of β-cyclodextrin-iodide, 17

A round bottom flask with a magnetic stirbar and a Schlenk adapter ischarged with 16, 15 equivalents of potassium iodide, and DMF. Theresulting mixture is heated at 80° C. for 3 hours, after which thereaction is allowed to cool to room temperature. The mixture is thenfiltered to remove the precipitate and the filtrate evaporated todryness and redissolved in water at 0° C. Tetrachloroethylene is addedand the resulting slurry stirred vigorously at 0° C. for 20 minutes. Thesolid is collected on a medium glass frit, triterated with acetone andstored over P₂O₅.

Example 20 Synthesis of β-cyclodextrin-thiol-PEG Appended Polymer, 18

Step 1: Synthesis of β-cyclodextrin-thiol (K. Fujita, et al., Bioorg.Chem., Vol. 11, p. 72 (1982) and K. Fujita, et al, Bioorg. Chem., Vol.11, p. 108 (1982))

A 50 mL round bottom flask with a magnetic stirbar and a Schlenk adapterwas charged with 1.00 g (0.776 mmol) of 16, 0.59 g (7.75 mmol) ofthiourea (Aldrich) and 7.8 mL of 0.1N NaOH solution. The resultingmixture was heated at 80° C. for 6 hours under nitrogen. Next, 0.62 g(15.5 mmol) of sodium hydroxide was added and the reaction mixture washeated at 80° C. under nitrogen for another hour. The reaction wasallowed to cool to room temperature before it was brought to pH 4.0 with10% HCl. The total solution volume was brought to 20 mL and then wascooled in an ice bath before 0.8 mL of tetrachloroethylene was added.The reaction mixture was stirred vigorously at 0° C. for 0.5 h beforethe precipitated solid was collected in a fine glass frit. The solid waspumped down overnight to yield 0.60 g (67%) of a white amorphous solid.

Step 2: A 100 mL round-bottom flask equipped with a magnetic stirbar anda reflux condensor was charged with 2.433 g (2.11 mmol) ofβ-cyclodextrin-thiol, prepared in Step 1, 0.650 g of functionalized PEG(PEG with pendant olefins, received from Yoshiyuki Koyama of OtsumaWomen's University, Tokyo, Japan) and 50 ml of dH₂O. The resultingmixture was heated at reflux for 12 hours, during which time theβ-cyclodextrin-thiol dissolved. The reaction mixture was allowed to coolto room temperature and precipitated solid was removed bycentrifugation. The supernatant was dialyzed against water in aSpectra/Por 7 MWCO 1,000 membrane. The solution was lyophilized to givean amorphous white solid.

Example 21 Synthesis of branched PEI-cyclodextrin polymer, 19

A 20 mL scintillation vial equipped with a magnetic stirbar is chargedwith branched PEI (25 kD, Aldrich) and 17. To this is added degassedsodium carbonate buffer. The resulting solution stirred at 80° C. for 4hours. The mixture is acidified with 0.1 N HCl and dialyzed withSpectra/Por MWCO 3,500 membrane for 2 days and lyophilized.

Example 21B Synthesis of PEI-Cyclodextrin Crosslinked Polymer

A branched PEI (Mw 1200, Aldrich) and difunctionalized cyclodextrinmonomer 2 (1 eq) are mixed in dry DMSO. The mixture is stirred at 80° C.for 4 days and then subjected to dialysis against water usingSpectra/Por MWCO 10,000 membrane for two days and lyophilized.

Example 22 Synthesis of Ad-PEG₃₄₀₀-Ad

240 mg of 1-aminoadamantane (1.60 mmol, Aldrich) and 288 mg ofPEG₃₄₀₀(SPA)₂ (0.085 mmol, Shearwater Polymers) was added to a glassvial equipped with a stirbar. To this was added 5 mL ofdicholoromethane, and the solution was stirred overnight. The next day,the solution was filtered to remove the n-hydroxysuccidimide byproductand the dichloromethane was removed in vacuo. The residue was dissolvedin water and centrifuged to remove excess 1-aminoadamantane. Thesupernatant was then dialyzed overnight in Pierce's Slide-A-Lyzer withMWCO=3500. The solution was then lyophilized to afford 248 mg of a whitefluffy solid of Ad-PEG₃₄₀₀-Ad.

Example 23 Synthesis of Ad-PEG₃₄₀₀-NH₂

347 mg of FMOC-PEG₃₄₀₀-NH₂ (0.110 mmol, Shearwater Polymers) and 155 mgof 1-aminoadamantane (1.0 mmol, Aldrich) was added to a glass vialequipped with a stirbar. To this was added 5 mL of dicholoromethane andthe resulting solution was stirred overnight. The next day, the solutionwas filtered to remove the n-hydroxysuccidimide byproduct and thedichloromethane was removed in vacuo. The residue was dissolved in waterand filtered to remove unreacted 1-aminoadamantane. The solution wasthen lyophilized to remove the water. The FMOC group was removed bydissolving the resulting solid in 20% piperidine in DMF for 20 minutes.The solvent was removed in vacuo and the residue redissolved in water.The solution was centrifuged to remove the undissolved FMOC and thendialyzed overnight in Pierce's Slide-A-Lyzer, MWCO 3500. The solutionwas then lyophilized to afford 219 mg of a white fluffy solid ofAd-PEG₃₄₀₀-NH₂.

Example 24 Adamantane-PEG₃₄₀₀-NH₂.(Ad-PEG₃₄₀₀-NH₂)

266 mg of FMOC-PEG₃₄₀₀-NHS (78.2 μmol, Shearwater Polymers, HuntsvilleAla.) were added to a glass vial equipped with a magnetic stirbar. 10eq. of 1-adamantane-methylamine (1.5 mmol, Aldrich) dissolved in 3 mL ofdichloromethane were then added and the solution stirred overnight atroom temperature. The solvent was removed in vacuo and water was addedto the remaining solution to dissolve the PEG product. The solution wascentrifuged at 20K rcf for 10 minutes, whereupon theadamantane-methylamine phase-separated as a denser liquid. The aqueousportion was collected and water removed in vacuo. The remaining viscousliquid was redissolved in 20% piperidine in DMF for FMOC deprotectionand stirred for 30 minutes at room temperature. The solvent was removedin vacuo, washed several times with DMF, redissolved in water, and runon an anionic exchange column to remove unreacted PEG. The firstfractions were collected and lyophilized to yield 222 mg of a white,fluffy powder (76% yield) of the desired product which was confirmed byMALDI-TOF analysis.

Example 25 Adamantane-PEG₃₄₀₀-Lactose (Ad-PEG₃₄₀₀-Lac)

60 mg of Ad-PEG₃₄₀₀-NH₂ (16.8 μmol), as prepared in Example 24, and 5.0eq of lactose-monosuccidimyl (50 mg, Pierce, Rockford, Ill.) were addedto a glass vial equipped with a stirbar. 2 mL of 50 mM NaHCO₃ was addedand the resulting solution stirred overnight. The reaction of the aminewas monitored by TNBS assay, that determines amine concentrations. Uponfull reaction of the amine (99% amine reacted), the solution wastransferred to a dialysis tubing (Slide-A-Lyzer, MWCO=3500, Pierce),dialyzed for 24 hours against water, and lyophilized to yield 65.1 mg ofa fluffy white powder (93% yield).

Example 26 Synthesis of Ad-PEG₅₀₀₀

279 mg of PEG₅₀₀₀-NHS (0.053 mmol, Shearwater Polymers) was added to aglass vial equipped with a stirbar. To this was added 46 μL of1-adamantane methylamine (0.42 mmol, Aldrich) dissolved in 3 mL ofdicholoromethane, and the solution was stirred overnight. The next day,the solution was filtered to remove the n-hydroxysuccidimide byproductand the dichloromethane was removed in vacuo. The residue was dissolvedin water and centrifuged. The excess 1-adamantane methylamine phaseseparated and the top aqueous phase was removed and dialyzed overnightin Pierce's Slide-A-Lyzer with MWCO=3500. The solution was thenlyophilized to afford 253 mg of a white fluffy solid of Ad-PEG₅₀₀₀. Theproduct was analyzed on a Beckman Gold HPLC system equipped with aRichards Scientific ELS detector and a C18 column and found to be pure(retention time of PEG₅₀₀₀-NHS: 10.7 min; retention time of product:12.0 min; acetonitrile/water gradient).

Alternative Synthesis Adamantane-PEG₅₀₀₀ (AD-PEG₅₀₀₀)

674 mg of PEG₅₀₀₀-NHS (135 μmol, Shearwater Polymers) were added to aglass vial equipped with a magnetic stirbar. 5 eq. of1-adamantane-methylamine (675 μmol, Aldrich) dissolved in 10 mL ofdichloromethane were then added and the solution stirred overnight atroom temperature. The solvent was removed in vacuo and water was addedto the remaining solution. The solution was centrifuged at 20K rcf for10 minutes, whereupon the adamantane-methylamine phase separated as adenser liquid. The aqueous portion was collected and dialyzed for 24hours (Slide-A-Lyzer, MWCO=3500) against water. The solution waslyophilized to yield 530 mg of a white, fluffy powder (75% yield,schematic of product shown below). The product was analyzed on a BeckmanGold HPLC system equipped with a Richards Scientific ELS detector and aC18 column and found to be pure (retention time of PEG₅₀₀₀-NHS: 10.7min; retention time of product: 12.0 min; acetonitrile/water gradient).AD-PEG₃₄₀₀ was synthesized using a similar protocol (56% yield; productconfirmed by Maldi-TOF analysis).

Example 27 Adamantane-(PEG₅₀₀₀)₂ (Ad-(PEG₅₀₀₀)₂)

315 mg of (PEG₅₀₀₀)₂-NHS (30 Shearwater Polymers) were added to a glassvial equipped with a magnetic stirbar. 10 eq. of1-adamantane-methylamine (300 μmol, Aldrich) dissolved in 3 mL of DCMwere then added and the solution stirred overnight at room temperature.The solvent was removed in vacuo and water was added to the remainingsolution to dissolve the PEG product. The solution was centrifuged at20K rcf for 10 minutes, whereupon the adamantane-methylamine phaseseparated as a denser liquid. The aqueous portion was collected anddialyzed for 24 hours (Slide-A-Lyzer, MWCO=3500) against water. Thesolution was lyophilized to yield 286 mg of a white, fluffy powder (91%yield).

Example 28 Adamantane-PEG₃₄₀₀-Fluorescein (Ad-PEG₃₄₀₀-FITC)

20 mg of Ad-PEG₃₄₀₀-NH₂ were dissolved in 3 mL of 0.1 M Na₂CO₃ in aglass vial equipped with a magnetic stirbar. To this solution were added3 eq of fluorescein isothiocyanate (FITC, Sigma) in DMSO (4 mg/mL, 1.6mL) and the resulting solution was stirred in the dark overnight beforetransferring to dialysis tubing (MWCO=3500) and dialyzing in the darkfor 48 hours against water. The solution was collected and lyophilizedto yield 23 mg of a yellow fluffy solid. PEG₃₄₀₀-FITC was synthesized asa control polymer from PEG₃₄₀₀-NH₂ (Shearwater Polymers) with the sameprotocol to yield 23 mg.

Example 29 Synthesis of GALA Peptide

The GALA peptide (sequence:W-E-A-A-L-A-E-A-L-A-E-A-L-A-E-H-L-A-E-A-L-A-E-A-L-E-A-L-A-A, MW 3032,SEQ ID NO: 1) was synthesized by the Biopolymer Synthesis Facility(Beckman Institute, California Institute of Technology) using anautomatic synthesizer. Before cleaving the peptide from the resin, onethird of the resin was set aside for adamantane conjugation. Analysis ofthe peptide by HPLC indicated greater than 95% purity.1-Adamantane-carboxylic acid (Aldrich) was conjugated to the N-terminalend of the GALA-peptide with DCC coupling chemistry. The resultingpeptide (GALA-Ad, MW 3194) was cleaved from the resin. Analysis of thepeptide by HPLC indicated greater than 90% purity. The identities of thepeptides were confirmed by MALDI-TOF analysis (Biopolymer AnalysisFacility, Beckman Institute, California Institute of Technology).

Example 30 Preparation of a Composition of the Invention Using GALAPeptide

Plasmids and oligonucleotides. Plasmid pGL3-CV (Promega, Madison, Wis.),containing the luciferase gene under the control of the SV40 promoter,was amplified by Esherichia Coli and purified using Qiagen'sEndotoxin-free Megaprep kit (Valencia, Calif.). Fluorescein-labeledoligonucleotides (FITC-oligos, 25-mer, 5′-FITC-ACT GCT TAC CAG GGATTTCAG TGC A-3′, SEQ ID NO: 2) were synthesized by the BiopolymerSynthesis Facility (California Institute of Technology).

Particle formation and characterization. Compositions of the inventionwere prepared by mixing an equal volume of 12 (dissolved in dH₂O) withDNA (0.1 mg/mL in dH₂O) at the appropriate charge ratios. The samevolume of GALA or GALA-Ad dissolved in 50 mM phosphate buffered saline(PBS, pH 7.2) was then added to the complexes. For example, withparticle characterization studies, 2 μg of plasmid DNA (20 μL) werecomplexed with 12 (20 mL) at a 5+/− charge ratio. 20 μL of GALAsolution, GALA-Ad solution or 50 mM PBS (for control samples) were thenadded to the complexes. The solution was then diluted with the additionof 1.2 mL dH₂O. The size and charge of particles were determined bydynamic light scattering and zeta potential measurements, respectively,using a ZetaPals dynamic light scattering detector (BrookhavenInstruments Corporation, Holtsville, N.Y.). The results, presented asmean±standard deviation of these measurements, are shown in FIG. 2. Thehydrodynamic diameter of 12/pGL3-CV compositions prepared at 5+/− chargeratio was measured by dynamic light scattering and found to be 260 mm. 2μg of plasmid DNA in 20 μL were mixed an equal volume of 12 at 5+/−charge ratio. Various ratios of GALA or GALA-Ad were then added to theparticles. Hydrodynamic diameter was determined by light scatteringmeasurements. Results are presented as mean±standard deviation of threemeasurements. The GALA peptide undergoes a transition from awater-soluble random coil conformation at pH 7.5 to a water-insolublehelix at pH 5. The GALA and adamantane-modified GALA (GALA-Ad) peptidewas dissolved in 50 mM PBS (pH 7.2) and added to the therapeuticcomposition at various peptide/cyclodextrin ratios. The mixture wasdiluted with dH₂O and particle sizes determined by dynamic lightscattering (FIG. 2). FIG. 2 shows the hydrodynamic diameter of GALA(deshed line) and GALA-Ad (solid line) modified polyplexes.

Results. Because the particle count rate remains the same for allconcentrations of peptide added, the addition of peptide does not appearto disrupt the compositions. The particle size profiles as a function ofGALA and GALA-Ad addition are very similar. The hydrodynamic diameterincreases from 250 mm (1% GALA or GALA-Ad) to 400 mm (10% GALA orGALA-Ad). As more peptide is added the particle size again decreases tothat of the unmodified therapeutic composition. The diameter returns toaround 250 nm with the addition of 30% or more GALA-Ad and 50% or moreGALA. See FIG. 2.

Example 31 Uptake of GALA-Modified Compositions to BHK-21 Cells

Cell Culture. BHK-21 cells were purchased from ATCC (Rockville, Md.) andHUH-7 cells were generously donated by Valigen (Newtown, Pa.). Both celllines were cultured in DMEM supplemented with 10% fetal bovine serum,100 units/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mLamphotericin in a humidified incubator operated at 37° C. and 5% CO₂ andpassaged every 4-5 days. Media and supplements were purchased from GibcoBRL (Gaithersburg, Md.).

Therapeutic composition uptake by cultured cells. BHK-21 cells wereplated in 6-well plates at 150,000 cells/well and incubated for 24 hoursat 37° C. 5 μg of FITC-oligo were complexed with 12 at a 5+/− chargeratio. After a 5 minute complexation time, 50 μL of GALA or GALA-Ad in50 mM of PBS (pH 7.2) were added to the complexes. Media was removedfrom the cells and cells washed with PBS. For transfection, 900 μL ofOptimem were added to each therapeutic composition solution and theentire solution transferred to the cells. The cells were incubated withthe transfection mixture for 5 hours before removing the media andwashing the cells twice with PBS. The cells were collected bytrypsinization and prepared for FACs analysis. Cells were washed twicein wash buffer (Hank's Balanced Salt solution containing DNase andMgCl₂) and resuspended in 500 μL FACS buffer (Hank's Balanced SaltSolution, 2.5 mg/mL bovine serum albumin, 10 μg/mL propidium iodide).FACS analysis was performed using a FACSCalibur flow cytometer (BectonDickinson, San Jose, Calif.) and CellQuest software. The results areshown as FIG. 4. As shown in FIG. 4 a-d, BHK-21 cells (4 a) weretransfected with 12/FITC-Oligo (4 b), 12/FITC-Oligo/50% GALA (4 c) and12/FITC-Oligo/50% GALA-Ad (4 d). Uptake was determined by flow cytometryanalysis. Data is presented as fluorescence profiles, with cell countnumber plotted along the y-axis and fluorescein fluorescence intensityplotted along the x-axis.

Example 32 Zeta Potential of Modified Complexes

2 μg of plasmid DNA in 20 μL were mixed with an equal volume of 12 at5+/− charge ratio. Various ratios of GALA or GALA-Ad were then added tothe particles at various peptide/CD ratios before dilution with dH₂O.Particle charge was determined by electrophoretic mobility measurementsand presented as particle zeta potential in mV. The particle charge of12/pGL3-CV compositions at 5+/− charge ratio was determined by zetapotential measurements and found to be +13 mV. The zeta potential of theparticles in the presence of the peptides was determined and presentedin FIG. 3 as mean±standard deviation of three measurements.

Results. Because the GALA peptide is an anionic peptide at pH 7.2(contains several glutamic acid residues), the association of GALA andGALA-Ad with the compositions decreases their zeta potential. Thecompositions become negatively charged by 30% GALA (−11 mV) or GALA-Ad(−23 mV). The zeta potential of GALA+ therapeutic composition solutionsplateaus at this point; adding more GALA only increases the zetapotential slightly (−15 mV at 150% GALA). However, the particles becomemore negatively charged with higher GALA-Ad concentrations, compositionswith the addition of 150% GALA-Ad have zeta potentials of −42 mV. SeeFIG. 3.

Example 33 DNA Delivery Efficiency of Compositions

HUH-7 Cells: A hepatoma cell line, HUH-7, was also transfected with12/FITC-Oligo at 5+/− charge ratio and 12/FITC Oligo/50% GALA-Adcompositions. DNA uptake was monitored as described for BHK-21 cells.The fluorescence profile for untransfected HUH-7 cells lies in the firstdecile (FIG. 5 a). FITC-Oligo was successfully delivered to 95% of HUH-7cells with 12 (FIG. 5 b). The addition of 50% GALA-Ad to thecompositions inhibits FITC-Oligo uptake by two orders of magnitude, asobserved with the BHK-21 cells (FIG. 5 c).

Example 34 Luciferase Transfection Efficiency of the InventionCompositions

The transfection ability of GALA and GALA-Ad modified compositions wasdetermined by delivery of a luciferase reporter gene to cultured cells.BHK-21 cells were plated in 24-well plates and transfected with 1 μg ofpGL-CV3 (a plasmid that contains the luciferase gene) complexed with 12at a charge ratio of 5+/− to form a particulate composite. Theseparticulate composites were modified with the addition of GALA orGALA-Ad at various peptide/cyclodextrin ratios. The cells were lysed 48hours after transfection and analyzed for luciferase activity, withresults, shown in FIG. 6, reported in relative light units (RLUs). Dataare reported as the mean±SD of three samples. Background=300 RLV.

Cells were successfully transfected with 12/pGL-CV3 compositions, withRLUs ˜1×10⁵. The addition of GALA did not have a large effect ontransfection efficiency. However, composition modification with GALA-Adgreatly inhibited transfection. The addition of 1% GALA increasedtransfection by two-fold to 2×10⁵ RLU, and 12/pGL-CV3/10% GALA alsoresulted in slightly higher transfections (1.5×10⁵ RLU). The addition of100% GALA decreased transfection by 50% to 5×10⁴ RLU.

Example 35 Toxicity of GALA and GALA-Ad Compositions

The toxicity of GALA and GALA-Ad modified compositions was determined bymeasuring the protein concentrations of the cell lysates obtained in thetransfection experiments. BHK-21 cells were transfected with 1 μg ofpGL-CV3 complexed with 12 at 5+/− charge ratio. Prior to transfection,various ratios of GALA and GALA-Ad were added to the complexes. Cellsurvival for transfections in the presence of GALA (solid bars) andGALA-Ad (white bars) was determined by assaying for total proteinconcentrations 48 hours after transfection and normalizing each samplewith protein levels for untransfected cells. The protein concentrationsare reported as the mean±SD of three replicates were averaged anddivided by the average protein concentration of cells transfected with12/pGL-CV3 compositions alone and reported as fraction cell survival(FIG. 7). The addition of GALA and GALA-Ad to the transfection solutionresulted in no observable toxicity to BHK-21 cells.

Example 36 Lactose-β-cyclodextrin-DMS copolymer 20(Lac-(3-cyclodextrin-DMS copolymer 20)

12 (20.5 mg, 3 μmol), 10 eq of ∀-lactose (21 mg, 60 μmol, Sigma), and18.6 mg of sodium cyanoborohydride (300 μmol) were added to a glassvial. 1 mL of borate buffer, pH 8.5 was added to the solids and theresulting solution was vortexed briefly before incubating in a 37° C.water bath for 30 hours. The solution was acidified to pH 6.0 with theaddition of 1M HCl and dialyzed against water for 24 hours. TNBS assayfor polymer amines revealed 87% conjugation. The structure of compound20.

Example 37 Lactose-(CH₂)₆β-cyclodextrin-DMS copolymer 21(Lac-C6-β-cyclodextrin-DMS copolymer 21)

12 (43.2 mg, 7.4 μmol) and 5.6 eq of mono(lactosylamido)mono(succinimidyl) suberate (50 mg, 84 μmol, Pierce) were added to aglass vial equipped with a magnetic stirbar and dissolved in 2 mL of 50mM NaHCO₃. The resulting solution was stirred overnight. The reactionwas followed by monitoring the disappearance of the polymer amineendgroups by TNBS assay, which revealed 90% conjugation. The solutionwas acidified to pH 5.0 by the addition of 1M HCl and resulting solutiondialyzed against water in Pierce MWCO 3500 Slide-A-Lyzer for 2 daysbefore lyophilization. A white, fluffy power was obtained in 70% yield.The structure of 21 is shown in FIG. 12.

Example 38 PEG₃₄₀₀-terminated β-cyclodextrin-DMS copolymer 22; Pre-DNAComplexation Pegylation

20.3 mg of 12 (3 μmol) and 10 eq of FMOC-PEG₃₄₀₀-NHS (190 mg, 60 μmol)were added to a glass vial equipped with a magnetic stirbar anddissolved in 1 mL of 50 mM NaHCO₃, pH 8.5. The solution was stirred inthe dark at room temperature for 20 hours and then lyophilized. Thesolid was dissolved in 0.5 mL of 20% piperidine in DMF and stirred for30 minutes for FMOC deprotection. The solvent was removed in vacuo andthe remaining viscous liquid dissolved in water and the pH brought below6.0 with 0.1 M HCl. The polymer was separated from unreacted PEG byanion exchange chromatography and lyophilized to yield a white fluffypowder. The structure of 22 is shown below.

Prep-DNA Complexation Pegylotion. Both 12 and 22 were mixed with plasmidDNA for particle size measurements. While βCDP6 12 condenses plasmid DNAto uniform particles with hydrodynamic diameter; 130 nm, pegylated 22 isunable to condense DNA. The presence of PEG at the polymer terminidisrupts DNA condensation.

Example 39 (Comparative) Post-DNA-Complexation Pegylation by Grafting

The procedure used was modified from Ogris et al., Gene Therapy, 6,595-605 (1999). 5 μg of pGL3-CV in 500 μL of dH₂O were mixed with anequal volume of PEI (in dH₂O) at a charge ratio of 3+/− or 6+/−. 12/DNAparticulate composites were prepared in the same manner at a chargeratio of 5+/−. Particle diameters of the particulate composites weremeasured by dynamic light scattering (DLS). After particulate compositeformation, PEG₅₀₀₀-SPA (10 mg/mL in DMF) was added to the solution mixedat room temperature for two hours. As a second stage after particle sizedetermination, 500 μL of PBS, pH 7.2, were added to the solution. Thesolution was incubated for 30 minutes at room temperature before finalparticle sizes were measured by DLS. See FIG. 8 for a schematicrepresentation.

In Stage 1, PEI/DNA or 12/DNA particulate composites were formed in 1.2mL dH₂O. The sizes of the particles were determined by dynamic lightscattering (DLS). PEG₅₀₀₀-SPA was added to the particulate compositesolutions Stage 2, and allowed to react with the polymer primary aminogroups for 1 hour. The sizes of the “pegylated” samples were measured byDLS. For Stage 3,600 μL of PBS, pH 7.2, were added to each sample totest the salt stability of pegylated particles. The particle sizes weredetermined 30 min after salt addition to determine the extent ofparticle aggregation.

PEI particulate composites were formulated 3+/− and 6+/− charge ratiosand 12/DNA particulate composites plexes were formulated at 5+/− chargeratio for Stage 1. PEG₅₀₀₀-SPA was added to PEI at 10:1 w/w according tothe procedure published by Ogris et al. Gene Therapy 6, 595-606, 1999.12 was pegylated with 100%, 150% and 200% PEG:amine (mol %). As acontrol, unreactive PEG was also added to 12 at 100%. The particlediameters at each stage are presented in the table of FIG. 9. The PEIparticulate composite increased slightly in size upon pegylation (58 nmto 65 nm for 3+/− charge ratio and 55 nm to 60 nm for 6+/− chargeratio). Pegylation protected the PEI particulate composites againstsalt-induced aggregation. While unmodified PEI particles increase indiameter to 800 nm after salt addition, pegylated PEI particulatecomposite increased slightly in size to 78 nm (for 6+/− charge ratio)and 115 nm (for 3+/− charge ratio).

The addition of 150% and 200% PEG₅₀₀₀-SPA to 12-based particulatecomposites resulted in particle disruption; particle counts dropdrastically and no consistent correlation function was observed.Pegylation of 12 likely prevents polymer/DNA binding. The particle sizeis maintained at 67 nm after pegylation with 100% PEG₅₀₀₀-SPA. However,monitoring of particle size as a function of time revealed that theparticles were disrupted for approximately 30 seconds after PEGaddition, after which the small particles were again observed.Therefore, the addition of 100% PEG₅₀₀₀-SPA may pegylate a fraction of12. Because the polymer 12 is added in excess with respect to the DNA(at a 5+/− charge ratio), the particles could then rearrange such thatthe unmodified polymers form polyplexes with the plasmid DNA while mostof the pegylated polymer remain free in solution. Salt addition to theseparticles results in particle aggregation (300 mm), although not to theextent of unmodified 12 particulate composite (700 nm). In summary,post-DNA-complexation pegylation to reaction with the polymer primaryamino groups is likely to be effective for high MW polymers with highcharge densities. However, reaction with 12, even post-DNA complexation,results in lack of salt stabilization at 100% PEG₅₀₀₀-SPA addition andparticle disruption with higher PEG₅₀₀₀-SPA concentrations.

Example 40 Post-DNA-Complexation Pegylation by Inclusion ComplexFormation

Using the procedure below, Adamantane-PEG (Ad-PEG) molecules were addedto solutions of preformed compositions at 100% adamantane tocyclodextrin (mol %). PBS was then added to the solutions and theparticle size monitored by DLS in 2 minute intervals. The results areshown in FIG. 10.

Procedure: 2 μg of pGL3-CV in 600 μL of dH₂O were mixed with an equalvolume of 12 (in dH₂O) at a charge ratio of 5+/−. The desired amount ofAd-PEG (10 mg/mL in dH₂O) was added and particle size determined by DLS.600 μL of PBS, pH 7.2, were added to the solution and particle sizemonitored in 2 minute intervals for 8 minutes.

The average diameter of unpegylated 12 particles increased from 58 nm to250 nm within 8 minutes after salt addition. The presence of free PEG insolution did not prevent aggregation (average diameter of 240 nm aftersalt addition). However, pegylation via inclusion complexes with linearAd-PEG molecules reduced particle aggregation in a length dependentmatter. 8 minutes after salt addition, particles pegylated withAd-PEG₃₄₀₀ aggregate to 210 nm in diameter while particles withAd-PEG₃₄₀₀-Lac aggregate to 200 nm. Particles pegylated with Ad-PEG₅₀₀₀only increase in diameter to 90 nm 8 minutes after salt addition and to160 nm 2 hours after salt addition. Modification with Ad-(PEG₅₀₀₀)₂ hada small effect on aggregation (particle diameter of 200 nm after saltaddition).

The stabilization also occurs in a PEG density-dependent manner (FIG.10A). The average particle diameter measured 10 minutes after saltaddition increases by 4.7-fold for unmodified polyplexes (58 nm to 272nm) but only 1.2-fold for polyplexes modified with the addition of 150%or 200% adamantane to cyclodextrin.

Example 41 Decreased Cellular Uptake due to Post-complexation Pegylation

Step 1: Transfection mixtures were prepared as follows: An equal volumeof cationic, 12 was added to 3 μg of FITC-Oligos (0.1 μg/μL in water) ata 3+/− charge ratio of polymer to DNA. To the complexes was added freePEG or Ad-PEG₅₀₀₀ (as prepared in Example 40) at a 1:1 PEG tocyclodextrin ratio.

Step 2. HUH-7 cells were plated at 3×10⁵ cells/well in 6 well plates andmaintained in 4 mLs of DMEM+10% FBS+Antibiotic/Antimycotic for 24 hours.After 24 hours, the cells were washed with PBS and 1 mL of Optimemcontaining the transfection mixtures of Step 1 was added to the cells.After a 15 minute incubation, the transfection media was removed, thecells were washed with PBS and 1 mL of Optimem was added to each well.The cells were incubated for another 30 minutes at 37° C. The cells werethen washed with Cell Scrub Buffer (Gene Therapy Systems) to removesurface-associated complexes and PBS and then detached from the wells bytrypsin treatment. The cells were then prepared and analyzed by FACSanalysis for FITC-Oligo uptake. The results are described in Table 1below. The modification of the complexes by Ad-PEG₅₀₀₀ decreases uptakeof the FITC-Oligo/polymer complexes.

TABLE 1 Sample Percent Transfected Cells alone  0% Cells + FITC-Oligo 0% Cells + Particulate composite + Free PEG 43% Cells + ModifiedAd-PEG₅₀₀₀ Particulate 27% composite

Example 42 12/Ad-PEG₃₄₀₀-FITC Composition Formation and Delivery toCultured Cells

BHK-21 cells were plated in 6-well plates at 200,000 cells/well andincubated for 24 hours at 37° C. 3 μg of oligo (0.1 mg/mL in dH₂O) werecomplexed with an equal volume of 12 (2 mg/mL in dH₂O) at a 5+/− chargeratio. After a 5 minute complexation time, 1.5 μL of PEG-FITC orAd-PEG-FITC (10 μg/mL in dH₂O) were added to the complexes. Media wasremoved from the cells and cells washed with PBS. For transfection, 940μL of Optimem were added to each therapeutic composition solution andthe entire solution transferred to the cells. The cells were incubatedwith the transfection mixture for 4 hours before removing the media,washing the cells with PBS, and adding in 4 mL of complete media. Thecells were incubated for another 24 hours at 37° C. before media wasremoved and cells washed twice with PBS. The cells were collected bytrypsinizatioh and prepared for FACs analysis. Cells were washed twicein wash buffer (Hank's Balanced Salt solution containing DNase andMgCl₂) and resuspended in 500 μL FACS buffer (Hank's Balanced SaltSolution, 2.5 mg/ml bovine serum albumin, 10 μg/mL propidium iodide).FACS analysis was performed using a FACSCalibur flow cytometer (BectonDickinson, San Jose, Calif.) and CellQuest software. FIG. 11 shows theresults.

Inclusion complex formation with AD-PEG₃₄₀₀-FITC resulted insubstantially increased fluorescein uptake over 12 incubated withAD-PEG₃₄₀₀-FITC (43% vs. 14%, FIG. 11). Free AD-PEG₃₄₀₀-FITC in themedia may be taken into the cell as part of the pinocytotic orendocytotic pathway. However, Ad-PEG₃₄₀₀-FITC is also able to entercells when complexed to 12. AD-PEG₃₄₀₀-FITC modification of 12particulate composites at low ratios (10%) is unlikely to inhibitinternalization. Rather, the 12 particulate composites bind readily tothe cell surface and co-delivers Ad-PEG₃₄₀₀-FITC to the cells as theyare internalized. The 12 particulate composites-assisted deliveryresults in higher fluorescein fluorescence observed in12/Ad-PEG₃₄₀₀-FITC transfected cells. This method can also be appliedfor the co-delivery of a small molecule therapeutic along with the geneof interest.

Example 43 Transfection of HU47 Cells

Luciferase Transfection. HUH-7 cells were plated in 24-well plates at50,000 cells/well and incubated for 24 hours at 37° C. 3 μg of pGL3-CVplasmid (0.1 mg/mL in dH₂O) were complexed with an equal volume of 12 or21 (See FIG. 13.) at various charge ratios. Media was removed from thecells prior to transfection and cells washed with PBS. 600 μL of Optimemwas added to each therapeutic composition to form a transfectionsolution of which 230 μL were added to each of 3 wells for 4 hours.After four hours, 800 μL of complete media was added to each well Mediawas changed 24 hours after transfection and cells were lysed in 50 μL ofCell Culture Lysis Buffer (Promega, Madison, Wis.) 48 hours aftertransfection. Luciferase activity was analyzed using Promega'sluciferase assay reagent. The results are shown in FIG. 13.

Example 44 Synthesis of Adamantane-derivatized PEI (Ad-PEI)

Polyethylenimine (PEI) and adamanetane carboxylic acid are mixed in dryCH₂Cl₂ and cooled to 0° C. DCC (1 equiv.), 1-hydroxybenzoyltriazole (1equiv.), and triethylamine (1 equiv.) are added to the mixture. Thesolution is warmed slowly to room temperature and stirred for 16 hours.The precipitate is removed by filtration and then the solvent is removedby vacuum. Water is added to the residual yellowish solid. Non-solublesolid is removed by centrifugation. The aqueous solution is carefullytransferred to a dialysis bag and dialyzed against water for 24 hours.The resulting PEI-CD is obtained after lyophilization.

Example 45 Synthesis of Cyclodextrin-PEG (CD-PEG)

PEG-Succinimidyl propionic acid (SPA) (Shearwater Polymers) andcyclodextrin-monoamine (1.2 equiv.) are dissolved in DMSO and stirredfor 24 hours at room temperature. The cyclodextrin-PEG product ispurified by dialysis.

Example 46 Formulation of Ad-PEI/DNA particulate composite andsubsequent modification with CD-PEG

1 μg of plasmid DNA (0.1 μg/μL in dH₂O) is mixed with Ad-PEI of Example42 at a 5+/− charge ratio. CD-PEG (dissolved in dH₂O) of Example 46 isthen added to the complex at the desired CD:Ad ratio.

Example 47 Stabilization by PEGylation: Formulation at HighConcentrations

4 μg of plasmid DNA was mixed with an equal volume of polymer mixture(containing cyclodextrin polymer 12 at a 2.5+/− charge ratio and, insome cases, Adamantane-PEG₅₀₀₀ or PEG₃₀₀₀ at 1 CD:1 PEG₅₀₀₀) at variousfinal DNA concentrations ranging from 0.1 mg/mL to 4 mg/mL (See FIG.14). Half of the solution was diluted with 1.2 mL of water and diameterdetermined by dynamic light scattering. The other half of the solutionwas passed through a Qiagen Qiaquick column to extract the DNA remainingin solution. The DNA concentration was determined by UV absorbance at8=260.

Results (FIGS. 15 and 16): Small and uniform particulate composites(diameter<100 nm) modified with Adamantane-PEG₅₀₀₀ can be formulated atconcentrations up to and including 4 mg DNA/mL without precipitation.Unmodified polyplexes form large particles (>300 nm) at concentrationsgreater than 0.2 mg/mL and extensive precipitation is observed (>50% DNAloss) at all formulation concentrations.

Example 48 Inhibition of Non-Specific Uptake by Polyplex SurfaceModification

BHK-21 cells were plated in 6-well plates. Cells were transfected with 3μg of FITC-Oligo (final concentration of transfection mixture: 0.05 mgDNA/mL) complexed with an equal volume of 12 at 2.5+/− charge ratio12/DNA. The particulate composites were then modified with the followinglinkers:

(SEQ ID NO: 3) Anionic Linker: WEAALAEALAEALAEAC (SEQ ID NO: 3)Ad-anionic linker: Ad- WEAALAEALAEALAEAC Ad-PEG Ad-PEG₅₀₀₀(SEQ ID NO: 3) Ad-anionic linker-PEG Ad-WEAALAEALAEALAEAC-PEG₅₀₀₀

1 mL of optimem was added to the transfection mixture and the totalsolution transferred to prewashed BHK-21 cells (rinsed with PBS) for 15minutes. Media was then removed and cells washed with CellScrub,trypsinized and prepared for FACs analysis.

Results: The inclusion guest (adamantane), spacer (anionic linker), andfunctional group (PEGD₅₀₀₀) work to modify 12/DNA particulate compositesand inhibit nonspecific uptake into cultured cells. See FIG. 17. Optimuminhibition is achieved with the combination of all three components.

Example 49 Galactose-Mediated Uptake into Hepatoma Cells

HepG2 cells were plated in 24-well plates at 50,000 cells/well. 1 μg ofpCMV-Luc was contacted with an equal volume of 12 and modified asindicated below. Modification with PEG-containing complexing agents wasdone at a 2:1 CD:PEG ratio, where CD represents the cycloclextins in 12.

12/pcMV-Luc Particulate Composite No Modification

(SEQ ID NO: 4) Glu-PEG-Pep-Ad Glucose-PEG₃₄₀₀-CAEAEAEAE-Ad,  2 CD: 1 PEG(SEQ ID NO: 4) Gal-PEG-Pep-Ad Galactose-PEG₃₄₀₀-CAEAEAEAE-Ad, 2 CD: 1 PEG (SEQ ID NO: 4) PEG-Pep-Ad PEG₅₀₀₀-CAEAEAEAE-Ad,  2 CD: 1 PEG

200 μL of Optimem was added to each transfection mixture and transferredto each well of cells. 4 hours after transfection, 800 μL of completemedia was added to each well. The media was removed, cells washed withPBS, and 1 mL of complete media added to each well 24 hours aftertransfection. 48 hours after transfection cells were washed with PBS,lysed and analyzed for luciferase activity. The described transfectionprocedure was also executed in the presence of 1 mM glucose or 1 mMgalactose as a competitive inhibitor.

Results: Particulate composites modified with Glu-PEG-Pep-Ad orPEG-Pep-Ad have a negative zeta potential and therefore do not readilytransfect cells. However, polyplexes modified with Gal-PEG-Pep-Ad showenhanced transfection that is inhibited in the presence of freegalactose, thus demonstrating galactose-mediated transfection intohepatoma cells. See FIG. 18.

Example 50 Synthesis of a Diadamantane Compound REFERENCE

-   Breslow, et al. JACS (1996) v118 p8495-8496.-   Zhang et al. JACS (1993) v115 p9353-9354

Anhydrous pyridine (5 mL) was put in a reactor containing a smallmagnetic stirbar and cooled in an ice bath. Methyldichlorophosphate (1.0mL) was added dropwise. The mixture was kept cold for another 15 minutesduring which a precipitate of N-methylpyridinium dichlorophophateformed. Adamantane ethanol dissolved in 5 mL of pyridine was added tothe reactor and the reactor sealed after the reaction mixture wasfrozen. The resulting mixture was stirred overnight at room temperature.The sealed reacted was then opened and the resulting mixture was pouredinto 10% sodium bicarbonate (50 mL). This resulting solution was thenevaporated in vacuo. 800 mL of water was added to the remaining solidand product extracted with 150 mL ether. The aqueous phase was acidifiedwith 2 N HCl to pH=1.4 and then extracted with 3×150 mL of CHCl₃:nBuOH(7:3). The organic layer was washed with water and the mixed solventswere evaporated in vacuo to form a solid phase. This solid wasrecrystallized with acetone/hexane, affording a white solid with 27%yield. Electrospray mass spectroscopy analysis revealed the pure,desired product.

Example 51 Synthesis of Diadamantane-PEG5000

Diadamantane-PEG₅₀₀₀

Dichloromethane was dried over CaH₂ at reflux overnight, then freshlydistilled before using it in the reaction. To a stirred solution ofPEG-epoxide (MW 5000) in freshly distilled dichloromethane (0.2 mL) wasadded slowly a solution of the bis(2-(1-adamantyl)ethyl phosphate (thediamantane compound described in Example 51) in 0.4 mL dichloromethane.The resulting solution was stirred at 35 degrees Celsius for 4 days. Thesolvent was removed in vacuo until dryness. 6 mL of water was added tothe solid formed, which generated a precipitate. The resulting mixturewas stirred for half an hour at room temperature and then centrifuged toeliminate the solid (unreacted diadamantane compound). The supernatantwas dialyzed overnight against a 3500MWCO membrane in water andlyophilized to dryness, which afforded a white solid with 99% yield.MaldiT of analysis revealed the desired product.

Example 52 Competitive Displacement Experiments between Ad-PEG₃₄₀₀ anddiadamantane-PEG₅₀₀₀

Competitive adsorption experiments were performed by adding a solutionof diAdPEG₅₀₀₀ to a pre-formed composition of AdPEG₃₄₀₀, polymer, andDNA. A salt solution was then added and particle size was measured as afunction of time. The initial was formed by addition of a 12 solution(16.6 μL water+2.61 μL of 12 at 5 mg/mL+2.37 μL of AdPEG3400 at 12.5mg/mL) to a DNA solution (20 μL of DNA at 0.1 mg/mL). Characteristics ofthis composition solution are as follows:

-   -   [DNA]=0.05 mg/mL    -   Molar ratio of AdPEG₃₄₀₀: CD=1:1    -   Charge ratio=3+/−    -   Total formulated volume=40 μL

This composition was allowed to incubate 10 minutes before the additionof di-AdPEG5K solution (10 mg/mL). The volume of this solution wasdetermined so that the molar ratio between diAdPEG5000 and AdPEG₃₄₀₀ was1:1, 1:2, 1:4, or 1:6. For example, when the ratio was 1:2, 2.38 μL ofdiAdPEG₅₀₀₀ solution was added.

After another 10 minutes of incubation, 1.2 mL of water was added todilute the so it could be read by the DLS instrument. Particle size wasmeasured for 10 minutes and then 600 uL of 1×PBS was quickly mixed intothe composition solution. Particle size was then observed each minutefor the next 30 minutes.

For comparison, two other composition solutions were formulated. In onecase, no diAdPEG₅₀₀₀ was added. In the other, no AdPEG₃₄₀₀ was added. Itcan be seen that under these conditions, the particulate composite sizeis not stabilized with the use of AdPEG₃₄₀₀. Salt causes the averageparticle diameter to increase from 70 nm to 350 nm over the course of 30minutes. However, diAdPEG₅₀₀₀ alone does show stabilization to salt.Particle size remains constant after the addition of salt solution. Thisis true even when the diAdPEG5K is present at ⅙ the amount of AdPEG3400.Results are shown in FIG. 19.

Example 53 pH Sensitive Adamantane-PEG Modifier

The association constant between an inclusion compound guest and hostdecreases when either the guest or host is charged. For example, theprotonated form (neutral form) of adamantanecarboxylic acid has anassociation constant ˜500,000, whereas the unprotonated (anionic) formof adamantanecarboxylic acid has an association constant ˜30,000. Thiscan be used to incorporate pH-sensitive behavior to a materialcontaining inclusion compounds. For example, a can be modified with anadamantarie-PEG (Ad-PEG) compound containing a secondary amine close tothe adamantane. The Ad-PEG compound would have high affinity for the atphysiological pH but would be more easily released at acidic pH, aswould be experienced inside cell endosomes. The facilitated unpackagingin the endosomes would promote DNA release have cellular internalizationof the polyplexes.

Synthesis of pH-Sensitive, hydrolysable Adamantane-PEG Modifiers

PEG5k-NH₂ (132 mg, 0.0264 mmol) was dissolved in water and cooled to 0°C. To the mixture was added NaOH solution (5N, 0.053 mL, 0.264 mmol, 10eq) and 1-adamantyl fluoroformate (52 mg, 0.264 mmol, 10 eq) THFsolution (3 mL). The mixture was stirred at such temperature for fiveminutes and then warmed up to room temperature and stirred for twohours. THF was removed under vacuum. The non-soluble solid was removedby centrifugation. The remaining aqueous solution was transferred toSpectra/Por MWCO 3,500 membrane and dialyzed against water for one day.The resulting Adamantane-carbamate-PEG5k (80 mg) was obtained afterlyophilization. The structure of this compound was confirmed by ¹H NMR,HPLC and MALDI TOF MS.

Synthesis of Hydrolysable Adamantane-Schiff Base-PEG

PEG5K-ALD and 1-adamantanemethylamine (1 eq) are mixed in methanol. Afew drops of formic acid is added the mixture as the catalyst for theformation of Schiff Base. The mixture is stirred at 60° C. for 12 hoursand then solvent is evaporated under vacuum. The mixture is dialyzed inwater to yield the desired Adamantane-Schiff Base-PEG5k.

Example 55 Synthesis of Adamantane-PEG-Transferrin (Ad-PEG-Tf), FIG.20 1. Transferring Coupling Via the Carbohydrate Groups Step 1:Synthesis of Ad-PEG-NH—NH₂

FMOC—NH-PEG₅₀₀₀-NHS (Shearwater Polymers, 0.2 mmol, 1 g) was added to around bottom flask equipped with a stir bar. To this was addedtert-butyl carbazate (Aldrich, 1.6 mmol, 0.2112 g) dissolved in 7 mL ofdichloromethane/Ethyl acetate (1:1). The resulting solution was stirredovernight at room temperature. The next day, the solvents were removedin vacuo. The FMOC group was removed by dissolving the resulting solidin 10 mL of 20% piperidine in dimethylformamide for 5 hours. The solventwas removed in vacuo and the residue was redissolved in water. Theresulting solution was centrifuged to remove the undissolved FMOC groupand then dialyzed overnight in Pierce's Slide-A-Lyser, 3500 MWCO. Thesolution was then lyophilized to afford 790 mg ofH₂N-PEG₅₀₀₀-NH—NH—CO—OtBu.

N-Hydroxysuccinimide (Aldrich, 0.24 mmol, 27.3 mg) andAdamantanecarboxylic acid (Aldrich, 0.39 mmol, 71.2 mg) were then addedto H₂N-PEG₅₀₀₀-NH—NH—CO—OtBu (2). (0.16 mmol, 790 mg) dissolved in 7 mLof dichloromethane. To this resulting solution was added 1,3-Dicyclohexylcarbodiimide (Aldrich, 1.6 mmol, 0.326 g) dissolved in 3mL of dichloromethane. The resulting solution was stirred overnight atroom temperature. The next day, the solid formed was filtrated on a fineglass frit and the filtrate was concentrated on a rotary evaporatorunder vacuum. The residue was dissolved in 10 mL of water andcentrifuged to remove the unreacted adamantanecarboxylic acid. Thesolvent was removed in vacuo and the residue was redissolved in 6 mL of4M HCl in dioxane in order to deprotect the t-Butoxycarbonyl group. Theresulting solution was stirred at room temperature for 4 hours. Thesolvent was then removed in vacuo and the residue was redissolved inwater. The resulting solution was dialyzed overnight in Pierce'sSlide-A-Lyser, 3500 MWCO and lyophilized to afford 635 mg ofAd-PEG₅₀₀₀-NH—NH₂.

Step 2: Transferrin-PEG-Ad Conjugate Synthesis

A solution of 100 mg (1.28 μmol) of Human Transferrin (iron poor)(Sigma-Aldrich) in 1 mL of a 30 mM sodium acetate buffer (pH 5) wassubjected to gel filtration on a Sephadex G-25 (Supelco) column. Theresulting 4 mL of solution containing Transferrin (monitoring: UVabsorption at 280 nm) was cooled to 0° C. and 80 μL of a 30 mM sodiumacetate buffer (pH 5) containing 4 mg (19 μmol) of sodium periodate wasadded. The mixture was kept in an ice bath and in the dark for 2 hours.For removal of the low molecular weight products an additional gelfiltration (Sephadex G-25, 30 mM sodium acetate buffer (pH 5)) wasperformed. This yielded a solution containing about 85 mg (1.09 μmol) ofoxidized Transferrin. The modified Transferrin solution was promptlyadded to a solution containing 54.5 mg (10.9 μmol) of Ad-PEG₅₀₀₀-NH—NH₂in 1 mL of 100 mM sodium acetate (pH 5). The resulting solution wasstirred overnight at room temperature. The pH was then brought to 7.5 byaddition of 1 M sodium bicarbonate and four portions of 9.5 mg (150μmol) of sodium cyanoborohydride each were added at 1 h intervals. After18 h, the PEGylated Transferrin was purified and concentrated using aCentricon YM-50,000 NMWI device (Millipore).

Step 3: Iron-Loading of Transferrin-PEG-Ad Synthesized by TransferrinOxidation

40 mg of apo-transferrin-based compound (apo-transferrin orapo-transferrin-PEG-Ad) was dissolved in 700 μL of dH₂O. To thissolution was added 200 μL of 5 mM Iron Citrate and 100 μL of 84 mg/mLNaHCO₃. This solution was allowed to stand for 2-3 hours and thendialyzed against PBS overnight. The iron-loading efficiency wascalculated by determining ratio of absorbance at 465 nm (from theoxidized iron) to the ratio of absorbance at 280 nm (from the tryptophanresidues in the protein) and normalizing to the A₄₆₅/A₂₈₀ ratio ofcommercially available holo-transferrin. The iron loading efficiency fortransferrin, transferrin in the oxidation buffer (sodium acetate pH 5)and freshly oxidized transferrin was determined and shown in FIG. 21.Oxidization of the transferrin reduces the iron loading efficiency ofthe protein.

Step 4: Binding Affinity of Transferrin-PEG-Ad (Synthesized byTransferrin Oxidation) to Transferrin Receptors on PC3 Cells

PC3 cells were incubated with 250 nM fluorescein-transferrin (FITC-TF)with various amounts of unlabeled transferrin and transferrin-PEG-Ad.FITC-hTF cell association was assessed by FACS analysis. Unlabeledtransferring competes very efficiently with the FITC-hTF, whereas thetransferring-PEG-AD competes very poorly with FITC-hTF, most likely dueto reduced affinity for the receptor. The results are shown in FIG. 22.

Example 56 Transferrin Coupling Via Lysine Groups, FIG. 23 Step 1:Synthesis of VS-PEG₃₄₀₀-Ad

Vinylsulfone-PEG₃₄₀₀-NHS (Shearwater Polymers, 0.147 mmol, 0.5 g) wasadded to a round bottom flask equipped with a stir bar and dissolved in5 mL of DMSO. To this was added Adamantanemethylamine (Aldrich, 0.147mmol, 0.0243 g). The resulting solution was stirred 1 h at roomtemperature. The solvent was removed in vacuo and the residue wasredissolved in water. The resulting mixture was dialyzed overnightagainst 1000 MWCO Membrane (Spectra Por). The solution was thenlyophilized to afford 0.49 g of Vinylsulfone-PEG₃₄₀₀-Ad.

Step 2: Transferrin-PEG-Ad (Tf-PEG-Ad) Conjugate Synthesis

A solution of 250 mg (3.21 μmol) of Human Transferrin (iron poor)(Sigma-Aldrich) in 10 mL of a 0.1 M sodium tetraborate buffer (pH 9.4)was added 109 mg (32.1 pimp of Vinylsulfone-PEG₃₄₀₀-Ad. The resultingsolution was stirred at room temperature for 2 hours. The PEGylatedTransferrin was purified from the unreacted Vinylsulfone-PEG₃₄₀₀-Adusing a Centricon YM-50,000 NMWI device (Millipore) and from theunreacted Transferrin using a Hydrophobic Interaction Column Butyl-650S(Tosoh Biosep) (confirmed by HPLC and MALI-TOF analysis).

Step 3: Iron-Loading of Transferrin-PEG-Ad Synthesized by Coupling ViaLysine Groups

Apo-transferrin and Tf-PEG-Ad were iron-loaded according to theprocedure described in Example 55. The extent of iron-loading wasquantified as described. The iron-loading efficiency of Tf-PEG-Adsynthesized by coupling via lysine groups was nearly 100%.

Example 57 Binding Affinity of Transferrin-PEG-Ad (Synthesized byCoupling Via Lysine Groups) to Transferrin Receptors on PC3 Cells

PC3 cells were plated in 6 well plates at 125,000 cells/ml. After 24hours, the cells were exposed to 250 nM FITC-Tf mixed with variousconcentrations of hTf, hTf-PEG-Ad (synthesized by oxidation of hTf),hTf-PEG-Ad (synthesized by VS-lysine reaction and purified) andhTf-(PEG-Ad)₂ (synthesized by VS-lysine reaction and purified). Uptakeafter 20 minutes exposure was determined by FACS. Unlike the Tf-PEG-ADsynthesized by transferrin oxidation, the Tf-PEG-Ad compoundssynthesized by lysine coupling competes effectively with the FITC-Tf forreceptors on the PC3 cell surfaces. Results are shown in FIG. 24.

Example 58 Zeta Potential of Tf-Modified Polyplexes

An equivolume aliquot of 12 was added to an aliquot of plasmid DNA (2 μgDNA, 0.1 mg/mL in water) at a 3+/− charge ratio to form the particulatecomposite. Holo-transferrin or holo-Tf-PEG-Ad (17 mg/mL in water) wasthen added to the particulate composite. The particles were diluted bythe addition of 1.2 mL of water and zeta potential determined bymeasurements on a ZetaPals dynamic light scattering instrument(Brookhaven Instruments). The results are shown in FIG. 25. Theunmodified holo-transferrin associates with the particulate compositesby electrostatic interactions. When 2 nmol Tf/μg DNA is added, theparticulate composites approach neutrality. The holo-Transferrin-PEG-Ad(designated Tf-PEG-Ad in FIG. 25) is likely to associate to theparticulate composites by both electrostatic and inclusion compoundinteractions. Therefore, there is a higher association of holo-Tf-PEG-Adwith the particles, as evidenced by the continued decrease in zetapotential of the modified particles with higher concentrations ofholo-Tf-PEG-Ad. At 2 nmol Tf/μg, particulate composites modified withholo-Tf-PEG-Ad are negatively charged (zeta potential˜−7 mV).

Example 59 Synthesis of AD-Phos-PEG₅₀₀₀-Galactose

Compound numbers below refer to the above scheme.

I. Synthesis of Adamantanephosphonic Acid. 2. Dibenzyl phosphite (0.712g, 2.71 mmol) was syringed into an argon protected1-adamantanemethylamine (0.493 g, 2.98 mmol) solution in dry CCl₄. Whiteprecipitate was observed almost immediately after addition of dibenzylphosphite. The solution was stirred for 12 hours. To the mixture wasadded CH₂Cl₂ (30 mL). The organic phase was washed with dilute acidicwater (pH=4) twice (2×40 mL). The organic phase was then dried withMgSO₄. The solvent was evaporated under vacuum. The resulting whitesolid was crystallized using a solvent mixture of CH₂Cl₂ and hexane.Needle crystals (0.69 g) 1 were obtained in 60% yield. The crystal wassubjected to hydrogenation using 10% Pd/C (200 mg) with hydrogen at apressure of 15 psi in ethanol (40 mL) for 16 hours. Catalyst was removedby filtration. The filtrate solvent was removed by vacuum. Quantitativeyield of 2 were obtained. The resulting compound 2 was used withoutfurther purification.

II. Synthesis of NH₂-PEG₅₀₀₀-Galactose 4. FMOC—NH-PEG₅₀₀₀-NHS(Shearwater, 760 mg, 0.152 mmol) was dissolved in DMSO (3.7 mL). To thissolution was added a solution of galactosamine (385 mg, 1.52 mmol) anddiisopropylethylamine (0.264 mL, 1.52 mmol) in DMSO (14 mL). Thesolution was stirred for 20 minutes and then dialyzed in water (4×4 L)using 3500 MWCO membrane (Spectra/Por 7, Spectrum Lab, Inc.) for 24hours. The solution was then lyophilized to afford 745 mgFMOC—NH-PEG₅₀₀₀-Galactose 3.3 was dissolved in DMF (12 mL) containingpiperidine (3 mL). The solution was stirred for 16 hours. DMF was thenremoved under high vacuum. To the resulting solid was added 40 mL water.The white solid was removed by centrifugation. The aqueous solution wasdialyzed in water (4×4 L) using 3500 MWCO membrane (Spectra/Por 7,Spectrum Lab, Inc.) for 24 hours. The solution was lyophilized to afford625 mg NH₂—PEG₅₀₀₀-Galactose 4.

III. Synthesis of Adamantane-Phos-PEG₅₀₀₀-Galactose 5.4 (63 mg, 0.013mmol) is dissolved in imidazole buffer solution (1 mL, 0.1 N, pH=6.5).To this solution is added a solution of 2 in CH₃CN (4 mL), and thenfollowed by the addition of1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Hydrochloride (EDC, 100mg, 40 eq.). The solution is stirred for 16 hours at room temperature.The solution is dialyzed in water (4×4 L) using 3500 MWCO membrane(Spectra/Por 7, Spectrum Lab, Inc.) and then lyophilized to yield 5.

Example 60 Synthesis of AD-Glu-Glu-PEG₅₀₀₀-Galactose

Compound numbers below refer to the above scheme.

I. Synthesis of H-Glu-Glu-Adamantane 7. H-Glu(Bn)-OH (3.55 g, 15 mmol)was dissolved in water (16 mL) containing sodium bicarbonate (1.26 g, 15mmol). To the mixture was added Z-Glu(Bn)-OSu (4.68 g, 10 mmol) in THF(30 mL). To the mixture was added another 30 mL THF, 20 mL CH₃CN andthen 2N NaOH 10 mL. The solution was stirred for 16 hours at roomtemperature. THF and CH₃CN was evaporated under high vacuum. To theaqueous mixture was added 1 N HCl to adjust the pH to 3. Precipitationwas observed. The mixture was extracted with chloroform (3×30 mL). Theorganic phase was dried with MgSO₄. MgSO₄ was removed by filtration.Organic solvent was evaporated to give white sticky solid 6. 6 was usedfor next step reaction without further purification.

6 (3.51 g, 6.1 mmol) was dissolved in dry THF (40 mL). To this solutionwas added 1-adamantanemethylamine (1.007 g, 6.1 mmol),1-hydroxybenzotriazole (0.93 g, 6.1 mmol), DCC (1.32 g, 6.4 mmol), anddiisopropylethylamine (1.06 mL, 6.1 mmol) under argon at 0° C. Themixture was then warmed to room temperature and stirred for overnight.Precipitate was filtered. THF was then removed under vacuum to yield ayellow solid. The yellow solid was crystallized in methanol to giveplate crystals 6 (2.1 g, 49%). 6 was then dissolved in 40 mL methanoland shaken in a hydrogenation apparatus in the presence of 200 mg 10%Pd/C under 25-30 psi hydrogen. Catalyst was filtered off after 24 hours.H-Glu-Glu-AD 7 was obtained in quantitative yield after methanol wasremoved under vacuum. 7 was used without further purification.

II. Synthesis of AD-Glu-Glu-PEG₅₀₀₀-Galactose 9.Vinylsulfone(VS)-PEG₅₀₀₀-NHS (Shearwater, 423 mg, 0.085 mmol) andgalactosamine (216 mg, 0.85 mmol) were added to a PBS solution (2.25 mL,1×, pH 7.2). The solution was stirred for 1 hour and then dialyzed inwater (4×4 L) using 3500 MWCO membrane (Spectra/Por 7, Spectrum Lab,Inc.) for 24 hours. The solution was then lyophilized. The product 8 wasanalyzed using MALDI-TOF and HPLC. 8 was dissolved in a borax buffersolution (6 mL, 0.1 N, pH 9.4). Compound 7 (121 mg) was dissolved inDMSO solution (2 mL) and then added to the polymer solution. The mixturewas stirred at 35° C. for 16 hours and then 50° C. for 7 hours. HPLC wasused to monitor this reaction. The polymer was dialyzed using 3500 MWCOmembrane and lyophilized to give 419 mg AD-Glu-Glu-PEG₅₀₀₀-Galactose 9in 90% yield.

Example 61 Synthesis of AD-Glu-Glu-PEG₅₀₀₀

Synthesis of AD-Glu-Glu-mPEG₅₀₀₀ 10. mPEG₅₀₀₀-SPA (Shearwater, 300 mg,0.06 mmol) and 7, Example 60, were dissolved in DMSO (2 mL) and CH₃CN (1mL). The mixture was stirred at room temperature for 24 hours. Thesolution was then dialyzed in water (4×4 L) using 3500 MWCO membrane(Spectra/Por 7, Spectrum Lab, Inc.) for 24 hours. The solution waslyophilized to give 276 mg Ad-Glu-Glu-mPEG₅₀₀₀ 10. 10 was confirmed byMALDI-TOF MS, HPLC, and ¹H NMR.

Example 62 Formulation of Transferrin and PEG-Modified Polyplexes

Polyplexes (polymer to DNA charge ratio of 3+/−) modified with Tf-PEG-AD(or Tf-(PEG-AD)₂) and PEG-AD (or PEG-Glu-Glu-AD) can be formulated asfollows. Equal volumes of all components are used. Tf-PEG-AD (orTf-(PEG-AD)₂) in water is added to a solution of 12 in water. To thismixed solution is added an aliquot of PEG-AD (or PEG-Glu-Glu-AD). Theternary mixture of polymers is then added to DNA solution. The solutionsare mixed gently by pipeting and particle size, zeta potential, and saltstability determined as described previously. The zeta potential of theparticles can be tuned by varying the relative ratios of Tf-PEG-AD (orTf-(PEG-AD)₂) vs. PEG-AD (or PEG-Glu-Glu-AD). Some examples of zetapotential variation and particle size as a function of particlemodification is shown in FIGS. 26, 27, and 28.

Example 63 Adamantane-anionicpeptide-PEG₃₄₀₀-galactose/glucose(AD-pep-PEG-gal/glu)

An anionic peptide (sequence: E-A-E-A-E-A-E-A-C, SEQ ID NO: 5) wassynthesized by the Biopolymer Synthesis Facility (Beckman Institute,California Institute of Technology) using an automatic synthesizer.Before cleaving the peptide from the resin, adamantane-carboxylic acid(ACA, Aldrich) was conjugated to the N-terminal end of the peptide withDCC coupling chemistry. The resulting peptide (ACA-E-A-E-A-E-A-E-A-C, MW1084) was cleaved from the resin and analyzed by Maldi-TOF.

Galactose- and glucose-PEG₃₄₀₀-vinyl sulfone (gal/glu-PEG₃₄₀₀-VS) wereprepared with approximately 95% yield by reacting NHS-PEG₃₄₀₀-VS(Shearwater Polymers) with 20 equivalents of glucosamine orgalactosamine (Sigma) in phosphate-buffered saline, pH 7.2 for two hoursat room temperature. The solution was dialyzed extensively against waterand then lyophilized. The thiols of the anionic peptide (twoequivalents) were reacted with galactose-PEG₃₄₀₀-VS orglucose-PEG₃₄₀₀-VS in 50 mM sodium borate buffer (pH 9.5) containing 10mM TCEP. The solution was acidified and the precipitated peptide(insoluble below pH 9.0) was removed by centrifugation. The supernatantwas collected, dialyzed extensively, and lyophilized. The desiredproducts were confirmed by Maldi-TOF analysis (schematic shown below).

Example 64 Synthesis of Naphthalene-PEG

500 mg of PEG₅₀₀₀-NHS (0.1 mmol, Shearwater Polymers) is added to aglass vial equipped with a stirbar. To this is added 146 μL of1-Naphthalenemethylamine (1 mmol, 10 eq, Aldrich) dissolved in 8 mL ofdicholoromethane, and the solution is stirred for 16 hours. The solventis then removed under vacuum. To the mixture is added 20 mL water.Non-soluble residue is removed by centrifugation. The aqueous solutionis dialyzed in Spectra/Por 3500 MWCO dialysis membrane for 24 hours. Thesolution is then lyophilized to afford a white fluffy solid ofNaphthalene-PEG₅₀₀₀. The product is analyzed using ¹H NMR, MALDI TOF MS,and reverse phase HPLC. Naphthalene-PEG₃₄₀₀ is synthesized using asimilar protocol (56% yield; product confirmed by Maldi-TOF analysis).

Example 65 Synthesis of Naphthalene-PEG5000-Galactose

Vinylsulfone(VS)-PEG₅₀₀₀-NHS (Shearwater, 423 mg, 0.085 mmol) andgalactosamine (216 mg, 0.85 mmol) were added to a PBS solution (2.25 mL,1×, pH 7.2). The solution was stirred for 1 hour and then dialyzed inwater (4×4 L) using 3500 MWCO membrane (Spectra/Por 7, Spectrum Lab,Inc.) for 24 hours. The solution was then lyophilized to yieldVinylsulfone-PEG₅₀₀₀-Galactose. The product was analyzed using MALDI-TOFand HPLC. Vinylsulfone-PEG₅₀₀₀-Galactose 300 mg (0.06 mmol) is dissolvedin a borax buffer solution (3 mL, 0.1 N, pH 9.4).1-Naphthalenemethylamine (8.8 μL, 0.06 mmol) is dissolved in DMSOsolution (3 mL) and then added to the polymer solution. The mixture isstirred at 55° C. for 36 hours. The polymer is dialyzed using 3500 MWCOmembrane and lyophilized to give Naphthalene-PEG₅₀₀₀-Galactose.

It should be understood that the foregoing discussion and examplesmerely present a detailed description of certain preferred embodiments.It will be apparent to those of ordinary skill in the art that variousmodifications and equivalents can be made without departing from thespirit and scope of the invention. All the patents, journal articles andother documents discussed or cited above are herein incorporated byreference in their entirety.

1-21. (canceled)
 22. An adamantane derivative of the formula:

wherein J is —NH—, —C(═O)NH—(CH₂)_(d)—, —NH—C(═O)—(CH₂)_(d)—, —CH₂SS—,—C(═O)O—(CH.₂)_(c)—O—P(═O)(O—(CH₂)_(c)-Ad)O—,

a peptide or polypeptide residue, or—NH(C═O)—CH(R¹)—NH—(C═O)—CH(R¹)—NH—; Ad is adamantyl; R₁ is—(CH₂)_(a)—CO₂H, an ester or salt thereof; or —(CH₂)_(a)—CONH₂; PEG is—O(CH₂CH₂O)_(z)—, where z varies from 2 to 500; L is H, —NH₂,—NH—(C═O)—(CH₂), —(C═O)—CH₂, —S(═O)₂—HC═CH₂—, —SS—, —C(═O)O— or acarbohydrate residue; a is 0 or 1; b is 0 or 1; d ranges from 0 to 6; eranges from 1 to 6; y is 0 or 1; and x is 0 or 1.